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What was the first gene(s) found to code for ncRNA

What was the first gene(s) found to code for ncRNA


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I could not find a source that states what the first gene found to code for ncRNA was. Someone told me, however, it was a gene that coded for either rRNA or tRNA. To this point in time I have had no other confirmation that this is true.


The first eukaryotic genes cloned were the 18S and 28S rRNA genes from the frog X. laevis as described here: http://www.genome.gov/25520303

And yes, they are definitely ncRNA genes.


Balanced gene regulation by an embryonic brain ncRNA is critical for adult hippocampal GABA circuitry

Genomic studies demonstrate that, although the majority of the mammalian genome is transcribed, only about 2% of these transcripts are code for proteins. We investigated how the long, polyadenylated Evf2 noncoding RNA regulates transcription of the homeodomain transcription factors DLX5 and DLX6 in the developing mouse forebrain. We found that, in developing ventral forebrain, Evf2 recruited DLX and MECP2 transcription factors to important DNA regulatory elements in the Dlx5/6 intergenic region and controlled Dlx5, Dlx6 and Gad1 expression through trans and cis-acting mechanisms. Evf2 mouse mutants had reduced numbers of GABAergic interneurons in early postnatal hippocampus and dentate gyrus. Although the numbers of GABAergic interneurons and Gad1 RNA levels returned to normal in Evf2 mutant adult hippocampus, reduced synaptic inhibition occurred. These results suggest that noncoding RNA–dependent balanced gene regulation in embryonic brain is critical for proper formation of GABA-dependent neuronal circuitry in adult brain.


Abstract

Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins. However, almost all means of gene identification assume that genes encode proteins, so even in the era of complete genome sequences, ncRNA genes have been effectively invisible. Recently, several different systematic screens have identified a surprisingly large number of new ncRNA genes. Non-coding RNAs seem to be particularly abundant in roles that require highly specific nucleic acid recognition without complex catalysis, such as in directing post-transcriptional regulation of gene expression or in guiding RNA modifications.


RESULTS

ERC production is controlled by several chromatin regulators

The involvement of chromatin in the control of both ribosomal ncRNAs transcription and rDNA recombination is particularly evident in the sir2Δ mutant. This strain shows a loss of silencing phenotype with ncRNA accumulation (Bryk et al., 1997 Li et al., 2006). Moreover, its ribosomal repeats are highly unstable, with frequent intra­chromatid recombination events generating ERCs (Gottlieb and Esposito 1989 Kaeberlein et al., 1999). The accumulation of ERCs in sir2Δ cells is one of the causes of their shortened lifespan (Kaeberlein et al., 1999). Considering that Sir2p is a histone deacetylase (Imai et al., 2000 Suka et al., 2002) and that the mutant's chromatin is hyperaccessible (Fritze et al., 1997 Cioci et al., 2002), we tried to clarify the role of histone acetylation and chromatin structure in recombination, in terms of ERCs formation, and in ncRNA transcription. To accomplish this task, we performed a biased screening, starting from the measure of ERCs on selected chromatin mutants (Figure 2).

FIGURE 2: Chromatin regulators control ERC formation. (A) Southern analysis of ERC species. DNA was isolated from the specified yeast strains or after 16 h treatment with 500 μM NAM, and probed with a radiolabeled rDNA sequence. ERCs are indicated by arrows. The differences in the migration of bands corresponding to ERCs are due to different durations of electrophoretic migration. (B) Quantification of ERC amount. Averages and SDs of at least three independent experiments are reported. Two-tailed t test was applied for statistical analysis. Asterisks indicate statistically relevant differences α = 0.05. The p values are as follows: sir2, p = 0.0419 NAM, p = 0.0086 rpd3, p = 0.0454 hst1, p = 0.1388 hst2, p = 0.6499 hst3, p = 0.0083 hst4, p = 0.1529 nhp6ab, p = 0.0474.

The HST1-4 genes code for NAD + -dependent histone deacetylases, which share a significant sequence identity with the SIR2 gene (Brachmann et al., 1995), giving rise to the S. cerevisiae sirtuin family. We measured ERCs on single mutants of SIR2 and HST1-4 genes and on a panel of other strains as follows. First, we analyzed WT cells treated with 500 μM nicotinamide (NAM), a sirtuin noncompetitive inhibitor that increases rDNA recombination and shortens replicative lifespan (Bitterman et al., 2002). Second, we extended the study to the mutant in a Zn + -dependent histone deacetylase, Rpd3p, unrelated to sirtuins. This enzyme antagonizes Sir2p in the establishment of silenced chromatin at telomeres, HM loci, and rDNA (Smith et al., 1999 Sun and Hampsey, 1999 Zhou et al., 2009 Ehrentraut et al., 2010), and a mutation in the RPD3 gene decreases the level of genomic recombination (Dora et al., 1999). Finally, we investigated the involvement of high-mobility-group Nhp6p proteins, which are chromatin architectural factors (Stillman, 2010). The double mutation of NHP6A and NHP6B genes results in a decreased amount of histones, which determines chromatin hyperaccessibility (Celona et al., 2011) and ERC accumulation, with a consequent shortened lifespan (Giavara et al., 2005).

To measure ERCs, we subjected whole genomic DNA purified from logarithmically growing cultures to electrophoresis and transferred it to a nitrocellulose filter by Southern blotting. The ERC species are detected as fast-migrating bands after hybridization with a radiolabeled probe (arrows in Figure 2A). Quantification of ERCs, obtained by normalizing the ERC level to the loaded genomic amount of each strain, is reported in Figure 2B. The graphs show the ERCs amount for each strain, or treatment, relative to the isogenic WT or untreated sample, indicated as 1. Data presented in this form allow a direct evaluation of fold changes in the different genetic backgrounds.

The sir2Δ strain shows increased ERCs levels, as previously described (Kaeberlein et al., 1999). In addition, NAM treatment of WT cells results in ERC accumulation, demonstrating that sirtuin catalytic activity is crucial to repressing recombination at rDNA. This finding was previously reported by measuring the rate loss of marker genes integrated at rDNA (Bitterman et al., 2002). Here we confirm the data specifically for intrachromatid recombination, which leads to ERC production.

Conversely, we observed almost complete absence of ERCs in cells lacking the histone deacetylase Rpd3p. As far as the HST1-4 sirtuins are concerned, we observed a significant increase in ERC amount in the hst3 mutant, whereas the extent of ERC formation was not affected by the other hst gene deletions. Finally, the nhp6ab double mutation enhances recombination at rDNA, as expected (Giavara et al., 2005).

Taken together, these data indicate that rDNA recombination is suppressed by HST3 and NHP6AB gene activity, besides that of the known SIR2. Moreover, global deacetylation activity on account of Rpd3p seems to be correlated with ERC production.

Altered ERC production corresponds to changes in ncRNA transcription

Previous studies indicated that the synthesis of ncRNAs at rDNA mediated by RNA polymerase II is related to instability of the ribosomal array. The most accepted model suggests that recombination regulation among the repeated units of the ribosomal cluster is achieved through ncRNA transcription (Kobayashi and Ganley, 2005).

To show whether ERC accumulation is stimulated by ncRNA transcription in our mutants, we analyzed the ribosomal ncRNA produced from the bidirectional promoter E-PRO (Figure 3A). In fact, E-PRO activity is directly involved in rDNA recombination, which is abolished when E-PRO is deleted or replaced with a unidirectional promoter (Kobayashi and Ganley, 2005). The NTS1-R ncRNA (Figure 3A) level was measured by reverse transcription-PCR (RT-PCR), using a strand-specific primer. PCR coamplification with an oligo pair for ACT1 was performed in order to normalize the data. Figure 3B reports the ncRNA level for each mutant and condition, expressed as ratio relative to the isogenic WT or untreated sample, respectively.

FIGURE 3: ERC formation is stimulated by ncRNA transcription. (A) Schematic map of the rDNA unit. Thin horizontal black arrows, ncRNAs produced from E-PRO and C-PRO promoters. Horizontal white arrow, oligo NTS1-R used in the strand-specific RT-PCR. (B) Quantification of the ncRNA expression level. The histograms indicate the averages and SDs from three independent experiments. Two-tailed t-test was applied for statistical analysis. Asterisks indicate statistically relevant differences α = 0.05. The p values are as follows: sir2, p = 0.0006 NAM, p = 0.002 rpd3, p = 0.0013 hst1, p = 0.0703 hst2, p = 0.1451 hst3, p = 0.0047 hst4, p = 0.125 nhp6ab, p = 0.0454.

SIR2 deletion leads to a twofold increase in the ncRNA expression, as previously reported (Li et al., 2006). In addition, the NAM treatment causes an up-regulation of ncRNA, even though the effect is less marked than in the sir2Δ mutant. The rpd3 mutant strain shows a significant reduction in ncRNA levels, consistent with the demonstrated role of this histone deacetylase in counteracting rDNA transcriptional silencing (Smith et al., 1999 Sun and Hampsey, 1999). The hst3 mutant accumulates ncRNA. On the contrary hst1Δ, hst2Δ, and hst4Δ strains show only slight differences in the production of this ncRNA transcript. Moreover, Pol II transcription of rDNA in the nhp6ab double mutant is threefold higher than its isogenic WT, providing the first evidence of the involvement of the high-mobility-group proteins in the regulation of ncRNA production.

Of interest, we observed strong correspondence between ERC production (Figure 2) and ncRNA expression profiles (Figure 3) in the analyzed chromatin mutants. In fact, mutants that produce more ERCs (sir2, hst3, and nhp6ab) also show elevated levels of ncRNA transcription. On the other hand, in the rpd3 mutant, rDNA recombination is partially suppressed and ncRNA expression significantly decreased.

In the mutants studied, the data are consistent with the model according to which ERC production is stimulated by ncRNA transcription (Kobayashi and Ganley, 2005).

Histone H4 acetylation correlates with ERC and ncRNA production

The foregoing data suggest a strong influence of the chromatin regulators (histone deacetylases and chromatin architectural factors) on the control of rDNA recombination mediated by ncRNA transcription. This last process was shown to be associated with changes in the histone acetylation pattern at rDNA (Cesarini et al., 2010).

To uncover the relationship between histone acetylation and transcription-induced recombination at rDNA, we analyzed the global acetylation level of histone H4 in all our strains. Indeed, the acetylation state of lysines within the N-terminal of this histone is crucial for the silencing control at telomeres, HM loci, and rDNA (Hecht et al., 1995 Braunstein et al., 1996 Imai et al., 2000 Hoppe et al., 2002 Zhou et al., 2009).

We assessed H4 histone acetylation through a chromatin immunoprecipitation (ChIP) approach (Rundlett et al., 1998). Chromatin extracts were immunoprecipitated with antibodies against the C-terminal region of histone H4 or the N-terminal acetylated form (at lysines 5, 8, 12, and 16). The immunoprecipitated DNA was analyzed by PCR using specific primers for six different rDNA regions: the 35S coding sequence (COD), the putative Pol I transcriptional enhancer (ENH), the E-PRO and C-PRO promoters, a region with highly positioned nucleosomes (NUC), and the ARS sequence (map details in Figures 1 and 4A). Considering that SIR2 mutations affect the total occupancy of histone H3 at rDNA (Li et al., 2006) and that the nhp6ab mutant shows a genome-wide reduction of nucleosome particles (Celona et al., 2011), we decided to normalize histone H4 acetylation to the whole H4 amount in all the strains. In addition, in the graphs, data are reported relative to the wild-type level of acetylation (blue line, Figure 4B).

FIGURE 4: The H4 acetylation profile is altered at the rDNA of histone deacetylases and mutants of high-mobility-group proteins. (A) Graphical representation of an rDNA unit. Horizontal thick black lines indicate the positions of PCR amplicons generated by the ChIP experiments (not to scale). (B) ChIP analysis of the histone H4 acetylation profile at rDNA. Chromatin from the specified strains was immunoprecipitated with antibodies against the C-terminal tail of histone H4 (H4 C-TERM) or against the histone H4 acetylated at lysines 5, 8, 12, and 16 (ACH4). The graphs show the acetylation enrichment, in the indicated rDNA regions, of the mutants relative to isogenic WT strain, reported as 1 (blue line). The lines show the average ratios and SD of technical replicates in a representative experiment out of two performed.

When the sir2Δ strain was compared with the WT, a strong increase in histone H4 acetylation throughout the nontranscribed spacer is observed, confirming previous observations (Bryk et al., 2002 Cioci et al., 2002). Similar results are obtained after inhibition of sirtuin catalytic activity by NAM. However, NAM increases histone acetylation to a lesser extent than does SIR2 deletion.

Unexpectedly, mutation of the histone deacetylase Rpd3p results in a significant decrease in the H4 acetylation level at rDNA. Similar results were reported for rpd3 mutants at telomeres specifically, this is true for H4K12 in yeast (Ehrentraut et al., 2010) and for several H4 lysine residues in Drosophila (Burgio et al., 2011). Our observation represents the first evidence of hypoacetylation at rDNA of rpd3 mutants.

Then we analyzed the H4 acetylation level in all hst1-4 mutants. We found that hst3 and hst4 strains show, on average, a 1.5-fold increase in H4 acetylation at all the analyzed regions (Figure 4), even though the deacetylation activity of these proteins has been reported only for histone H3 lysine 56 (Xu et al., 2007 Yang et al., 2008). For hst1 and hst2 mutants we could not detect any significant variation relative to the isogenic WT strain (unpublished data).

An increase of histone H4 acetylation in the NTS is also evident for mutant strains lacking the NHP6A and NHP6B genes, suggesting a misregulation in the balance of acetylation/deacetylation in mutants of the high-mobility-group proteins.

To verify whether the alteration in histone acetylation involves other silenced regions, we extended the ChIP analysis to the telomere of the right arm of chromosome VI (Supplemental Figure S1B). We found changes in the acetylation patterns for sir2Δ, rpd3Δ, and NAM-treated cells, similar to what happens at rDNA. Conversely, in hst3 and nhp6ab mutants, no alteration in the level of histone H4 acetylation is observed at telomeres, indicating that their effect on H4 acetylation is restricted to the ribosomal locus (Supplemental Figure S1B).

Strikingly, the data reported in Figures 2–4 show a clear association between ERC production, ncRNA transcription, and histone H4 acetylation among the strains analyzed. In fact, mutants with alterations in ERC and ncRNA levels also show a corresponding change in the histone H4 acetylation. However, the analyses performed so far do not allow us to discriminate which lysine residues account for the observed differences, because of the multiple recognition specificity (lysines 5, 8, 12, and 16) of the antibody used in the ChIP assay.

H4K16 acetylation overlaps with H4 global acetylation in chromatin mutants

We then decided to measure the acetylation level of the H4K16 residue in all mutants for the following reasons: 1) H4K16 acetylation has been found to be associated with Pol II transcription in euchromatic regions in all eukaryotes (Millar et al., 2006) 2) its deacetylation is crucial for transcriptional silencing at all three silent loci in yeast (Hecht et al., 1995 Braunstein et al., 1996 Hoppe et al., 2002) and 3) H4K16 is the main deacetylation target of Sir2p, the silencing mediator (Imai et al., 2000). Considering the changes in ncRNA transcription in the mutants studied, it is conceivable that acetylation of this residue plays a role in the alterations of the silencing process at rDNA.

When ChIP extracts were immunoprecipitated with antibodies against the acetylated form of H4K16 (H4K16-ac), the overall result, obtained after normalization to the total amount of histone H4, was similar to that shown for the globally acetylated form of H4 (Figure 5 also see ACH4 reported in Figure 4).

FIGURE 5: The alterations in H4 Lys-16 acetylation correlate with the observed changes in global H4 acetylation. (A) Schematic map of the rDNA repeat. The fragments amplified in the ChIP analyses are shown. (B) ChIP analysis of the histone H4 Lys-16 acetylation at rDNA. As in Figure 4B, except that the antibodies used were against the C-terminal tail of histone H4 (H4 C-TERM) or against H4 acetylated at Lys-16 (H4K16-ac).

By comparing the acetylation profiles of the sir2 mutant, obtained with αH4K16-ac and αACH4 antibodies, we observed that H4K16 acetylation increases to a lesser extent. Possibly this is due to the fact that H4K16 is not the only residue whose acetylation increases in the sir2 mutant (Braunstein et al., 1996). The NAM treatment appeared to inhibit with different efficiency H4K16-ac and ACH4. In fact, NAM-treated cells show the same H4K16 acetylation extent as the sir2∆ strain, whereas this is not evident for the global H4 acetylation.

According to the previous results (Figure 4), the rpd3 mutant shows a sharp decrease in the H4K16 acetylation in all the rDNA regions studied. Acetylation of the hst3 and hst4 mutants seems to be slightly affected, whereas the effect of nhp6 mutation is, again, higher. Moreover, Hst3p, Hst4p, and Nhp6p seem to influence H4K16 acetylation specifically at rDNA, as confirmed by the fact that the acetylation extent of a subtelomeric region of the telomere VI is not altered in the mutant strains (Supplemental Figure S1C).

Because Hst3p and Hst4p deacetylases are known to act on H3K56 and Rpd3p is specific for H4K12 (among other lysines), we evaluated the acetylation extent of these residues (Supplemental Figure S2). We found a slight decrease in H4K12-ac and a small increase in H3K56-ac at the rDNA of rpd3 and hst3 mutants, respectively. Conversely, no significant changes were observed when the analysis was extended to the telomeres of these strains (Supplemental Figure S2).

Taken together, the data highlight a clear selective H4K16 acetylation accumulation (Figure 5) at the ribosomal locus in those mutants where both ncRNA and ERC amounts are increased (sir2, nhp6ab, and, partially, hst3). Correspondingly, in the rpd3 mutant, hypoacetylation of H4K16 seems to be related to a decrease in both ERC and ncRNA production (Figures 2, 3, and 5).

H4K16 acetylation controls ERC accumulation and ncRNA transcription

To unambiguously attribute a role to H4K16 acetylation as a key regulator of recombination and Pol II transcription at rDNA, we used yeast strains bearing substitutions of H4K16 to arginine (H4K16R) or glutamine (H4K16Q). These two modifications mimic the unacetylated or acetylated state of the residue, respectively. H4K16R and H4K16Q mutations were previously shown to alter silencing in yeast (Hecht et al., 1995 Meijsing and Ehrenhofer-Murray, 2001 Hoppe et al., 2002).

In Figure 6 we report results on DNA recombination, measured as ERC accumulation (Figure 6A), and Pol II transcription, measured as ncRNA production (Figure 6B). H4K16Q, H4K16R, and their isogenic WT strains were grown to the exponential phase and ERC species analyzed as described in Figure 2. The H4K16Q strain shows a twofold increase in ERC content relative to the isogenic WT. In contrast, the H4K16R mutation maintains the amount of ERC at the level of WT (Figure 6A).

FIGURE 6: Deacetylation of H4K16 is required for the suppression of ERC formation and ncRNA transcription. (A) Substitution of histone H4 Lys-16 with glutamine increases ERC production. Quantification of ERC amount produced by yeast strains bearing substitutions of H4K16 to glutamine, H4K16Q (left), or to arginine, H4K16R (right). ERC species were quantified and normalized as described in Figure 2 (n > 3 ±SD). Two-tailed t test was applied for statistical analysis. Asterisk indicates the statistically relevant difference, α = 0.05, between WT (PKY501) and H4K16Q (p = 0.0204) strains. (B) The H4K16Q mutant up-regulates ncRNA transcription at rDNA. Measure of the E-PRO–derived ncRNA expression level of H4K16Q (left), H4K16R (right), and the corresponding isogenic WT as in Figure 3 (n = 3 ±SD). Asterisk indicates the statistically relevant difference, α = 0.05, between WT (PKY501) and H4K16Q (p = 0.0264) strains.

We then measured the ncRNA expression level, derived from the E-PRO promoter, as in Figure 3. Figure 6B shows how, even in this case, H4K16Q mutation causes loss of silencing at rDNA, with an increased steady-state level of ncRNA, whereas the H4K16R strain seems to produce ncRNA at a similar rate as the WT.

Overall these data indicate that the replacement of the positively charged lysine residue 16 with a neutral one is sufficient to induce a coordinated deregulation of both ncRNA transcription and recombination, thereby suggesting a pivotal role of H4K16 acetylation for the control of TAR at rDNA.

The nhp6ab mutation affects SIR2 expression

The results reported here show that H4 Lys-16 acetylation is sufficient to control TAR at rDNA, because in H4K16Q mutants both processes are affected. In addition, in sir2, rpd3, hst3, and nhp6ab mutant strains a clear relationship between the acetylation state of H4K16 and recombination regulation by ncRNA is observed.

A direct interpretation of the mechanism underlying these phenotypes is possible only for the sir2Δ strain, since only Sir2p deacetylates H4K16 both in vitro and in vivo. A possible explanation for the observed variations in H4K16 acetylation in the other mutants could be defective recruitment of Sir2p at the ribosomal array.

We tested this hypothesis by measuring Sir2p binding at rDNA by ChIP analysis. Chromatin extracts of rpd3, hst3, and nhp6ab mutants were immunoprecipitated with αSir2 antibodies, and DNA was amplified in two regions, ENH and C-PRO, previously shown to be specifically bound by Sir2p (Huang and Moazed, 2003). We found that the Sir2p enrichment at rDNA of the nhp6ab mutant drops relative to the isogenic WT (Figure 7A). Conversely, rpd3 and hst3 mutants did not show significant changes in the level of Sir2p recruitment (Supplemental Figure S3). The reduction observed in the nhp6ab strain involves both regions to a very similar extent.

FIGURE 7: The nhp6ab mutant has reduced SIR2 expression. (A) The nhp6ab double mutant leads to the loss of Sir2p from the rDNA locus. WT and nhp6ab strains were subjected to ChIP analysis with antibodies against Sir2p. The recovered DNA was amplified in two regions known to bind Sir2p—the enhancer (ENH) and the C-PRO promoter. (B) Sir2 mRNA expression is reduced in nhp6ab cells. The graph reports the Sir2 mRNA levels of nhp6ab and WT strains measured by RT-PCR (n = 3 ±SD). (C) The nhp6ab mutant cells have a decreased Sir2 protein level. Protein extracts from three independent cell cultures of WT (left) or nhp6ab (right) cells were analyzed by Western blot with anti-Sir2 and anti–α-tubulin antibodies.

We hypothesized that the proportional decline of Sir2p binding at C-PRO and ENH could reflect a general reduction in Sir2p availability in nhp6ab mutants. To verify this hypothesis, we measured SIR2 mRNA expression and protein level by RT-PCR and Western blot (Figure 7). Compared to the WT, the mutant shows a twofold reduction of SIR2 mRNA levels (Figure 7B), and the Sir2 protein amount is even more reduced (Figure 7C).

To verify whether the effects of nhp6ab mutation are limited to the ribosomal locus, we also measured the Sir2p recruitment at telomere VI R. We found only a slight decrease in Sir2p binding at the telomere (Supplemental Figure S4A). Indeed, telomeric silencing of nhp6ab is unaffected, indicating that the observed decrease is not influential (Supplemental Figure S4B).

SIR2 overexpression restores most of the WT phenotypes in the nhp6ab mutant

The increase in ncRNA transcription, ERC accumulation, and H4 hyperacetylation in the nhp6ab mutant may all be explained by the observed decrease in the SIR2 gene product (Figure 7). If this were the case, the ectopic expression of the SIR2 gene should restore rDNA silencing, the control of recombination, and the WT level of acetylation in the nhp6ab strain.

Thus we transformed nhp6ab and WT cells with the pAR44 plasmid, in which SIR2 is under the control of the GAL10 promoter (Holmes et al., 1997). These transformants were compared with cells transformed with an empty vector for ncRNA production (Figure 8A), H4 histone acetylation (Figure 8, B and C), and ERC formation (Figure 8D). All the strains were grown in galactose to induce SIR2 expression.

FIGURE 8: SIR2 overexpression restores the WT level of ncRNA transcription and H4 acetylation in the nhp6ab mutant. (A) SIR2 overexpression in the nhp6ab mutant restores the levels of ncRNA transcription. WT and nhp6ab cells were transformed with the pAR44 plasmid (+) or with the empty vector (–). Quantification of ncRNA expression was performed as described in Figure 3 (n ≥ 4 ±SD). Two-tailed t test was applied for statistical analysis. Asterisks indicate the statistically relevant differences between nhp6ab cells with or without SIR2 overexpression (α = 0.01 p = 0.0015) or between WT cells with or without SIR2 overexpression (α = 0.01 p = 0.009). (B) SIR2 overexpression rescues the altered histone H4 acetylation of the nhp6ab mutant. Cells overexpressing (+) or not (–) SIR2, were grown in galactose and processed for ChIP analysis with antibodies against the C-terminal tail of histone H4 or against the acetylated histone H4 (ACH4). Quantification details and positions of PCR fragments are as in Figure 4. (C) The increased H4K16 acetylation of nhp6ab drops to the WT level after SIR2 overexpression. As in B, except that the antibodies were anti–acetyl histone H4-Lys-16 (H4K16-ac) and anti–H4 C-terminal tail (H4 C-TERM). (D) SIR2 overexpression does not reduce the ERC levels in the nhp6ab strain. DNA from cells containing the PAR44 plasmid (+) or the empty vector (–) was extracted and processed for ERC analysis as described in Figure 2 (n = 3 ±SD).

In this case, the ncRNA expression value of WT cells transformed with the empty plasmid was used as normalizer and is reported as 1 in the graph in Figure 8A. We observed a significant reduction of ncRNA expression for the nhp6ab mutant overexpressing SIR2 when compared with the same strain without the ectopic expression of SIR2. WT cells transformed, as control, with pAR44 also show a significant reduction of ncRNA relative to the corresponding WT strain without SIR2 overexpression. A parallel analysis was performed on cells grown in the repressive glucose-containing medium. However, because of the leakiness of the GAL10 promoter (Lange et al. 2000), a conspicuous SIR2 expression was detected, and the restoration of ncRNA silencing in nhp6ab mutant was observed (unpublished data).

The phenotype rescue analysis was extended to the global H4 acetylation levels (ACH4) in the different rDNA regions of the same strains studied earlier (Figure 8B). The measure of H4 acetylation was performed by ChIP, as reported in Figure 4. When WT cells containing pAR44 are compared with WT cells transformed with the empty plasmid, a similar acetylation profile is observed in most of the regions analyzed. Conversely, a sharp decrease of H4 acetylation is evident in the nhp6ab mutant that overexpresses SIR2 relative to nhp6ab cells without SIR2 ectopic expression. Indeed, SIR2 overexpression makes the H4 acetylation levels almost identical in WT and nhp6ab cells.

We further analyzed the specific acetylation of histone H4 at the Lys-16 residue (H4K16-ac). The acetylation profiles (Figure 8C) are comparable to those observed with αACH4 antibodies. This further demonstrates that the H4K16 acetylation overlaps with H4 global acetylation at the rDNA locus.

The ncRNA expression and the H4 acetylation levels obtained with cultures using glucose as carbon source (Figures 3–5) show similar profiles, but different absolute values, compared with those observed when cells are grown in galactose (Figure 8). Given that Sir2p is a NAD + -dependent enzyme, and given its pivotal role in these processes, changes due to the metabolic alterations are conceivable.

The same strains were also analyzed for ERC production (Figure 8D). However, for this phenotype, SIR2 overexpression did not restore the WT level of recombination.


NcRNA transcription makes its mark

A recently recognized strategy for gene regulation involves transcription of a non-coding RNA (ncRNA) transcript that overlaps the gene targeted for regulation. In many cases, it seems that it is the act of transcription itself rather than the ncRNA transcript that mediates regulation. A paper in this issue of the EMBO Journal shows one mechanism by which these transcription events regulate transcription elongating RNA polymerases direct a set of regulatory histone modifications that modulate expression of an overlapping gene.

A major surprise of the past few years has been the discovery of significant transcription activity across entire eukaryotic genomes, showing a large class of ncRNAs that are often rapidly degraded ( Yazgan and Krebs, 2007 ). In a number of cases, these ncRNAs have been found to regulate gene expression. Most of these regulatory ncRNAs function through RNAi-mediated pathways of gene repression. However, some ncRNAs regulate gene expression in cis. In these cases, the act of transcription itself, rather than the RNA product of transcription, mediates regulation of an overlapping gene.

The proposed mechanisms for regulation in cis include promoter occlusion or transcriptional interference by RNA polymerases transcribing ncRNAs ( Yazgan and Krebs, 2007 ). Other genes show regulated transcription start-site choice from a single promoter, giving rise to either a coding transcript or an ncRNA ( Jenks et al, 2008 Kuehner and Brow, 2008 ). A cryptic promoter that lies at the 3′ end of the PHO5 gene and drives an antisense transcript is required for the normal kinetics of PHO5 activation ( Uhler et al, 2007 ). Transcription of a series of ncRNAs upstream of the Schizosaccharomyces pombe fbp1+ promoter is required for its induction when cells are shifted to inducing conditions ( Hirota et al, 2008 ). Passage of RNA polymerase II through the fbp1+ promoter during transcription of these ncRNAs promotes the formation of open chromatin, allowing the transcription factor access to the fbp1+ promoter during induction.

At present, reports by Houseley et al (2008 ) and by Pinskaya et al (2009 ) provide compelling evidence that transcription of ncRNAs influences post-translational modifications of histones that facilitate the repression of overlapping genes.

Chromatin immunoprecipitation (ChIP) experiments carried out by Houseley et al showed a surprising pattern of Set1-dependent histone H3K4 trimethylation across the well-characterized GAL1–10 gene locus (Figure 1). A significant peak of this histone methylation mark, normally associated with the 5′ end of transcribed genes, was found within the 3′ end of GAL10 when cells were grown in glucose medium (GAL1-10 repressing conditions). These observations led Houseley et al to identify and characterize a set of ncRNAs that are transcribed from the 3′ end of GAL10 across the promoter region shared by the divergent GAL1 and GAL10 genes, which they named GAL10 ncRNAs.

Pinskaya et al observed that cells lacking Set1 induced GAL1–10 expression more rapidly than wild-type cells when cells were shifted to galactose medium, although the final, fully induced levels of GAL mRNA were unchanged. The increased expression of GAL1–10 in set1 cells correlated with TBP occupancy at the GAL1–10 promoter, suggesting that Set1 regulates transcription initiation at GAL1–10. Furthermore, an H3K4A mutant showed a similar induction phenotype, indicating a role for H3K4 methylation in GAL1–10 induction. Subsequent experiments identified a set of ncRNA transcripts similar to those reported by Houseley et al, which they named GAL1ucut (GAL1 upstream cryptic unstable transcripts).

Both groups mapped the GAL1ucut promoter to a location in the 3′ end of GAL10 near a pair of binding sites for the Reb1 transcription factor. Mutation of REB1, or of the Reb1 sites in GAL10, abolished GAL1ucut expression. Furthermore, both groups found an inverse relationship between GAL1ucut and GAL1–10 expression. GAL1ucut is expressed under conditions that repress GAL1–10, and as GAL1–10 is induced GAL1ucut declines. Curiously, Houseley et al did not observe an effect of GAL1ucut on GAL1–10 expression when cells were shifted to a medium with high levels of galactose. Rather, they observed that GAL1ucut antagonized the induction kinetics and final levels of GAL1–10 in a medium with low levels of both glucose and galactose. The basis for the difference in observations between the groups is not obvious, but both agree that GAL1ucut is used to attenuate GAL1–10 expression.

Both groups argue that GAL1ucut acts in cis. First, Houseley et al formed a heterozygous diploid yeast strain in which one of the two GAL1–10 loci lacked the GAL1ucut promoter. They observed no attenuation of GAL1–10 expression in this strain. Second, both groups found that GAL1ucut RNA was stabilized by mutations affecting RNA degradation pathways used to target ncRNA, and Pinskaya et al showed that this stabilization had no effect on GAL1–10 induction.

Earlier work has shown that Rpd3S histone-deacetylase complex is recruited to the body of protein-coding genes by H3K36-methylated nucleosomes ( Lee and Shilatifard, 2007 ). This serves to inhibit intragenic transcription from cryptic promoters that might otherwise be activated by the passage of transcription elongation complexes. Houseley et al observed that histone modifications, which are the hallmarks of this Rpd3S-mediated intragenic repression mechanism, methylation of histone H3K36 and subsequent histone deacetylation, were found across the repressed GAL1–10 locus. Furthermore, these marks were dependent on GAL1ucut transcription, and deletion of the Eaf3 subunit of the Rpd3S complex relieved glucose repression to a level similar to that observed when the GAL1ucut promoter was deleted.

Pinskaya et al also found a role for Rpd3S in GAL1ucut function. As H3K4-methylated histones can be recognized by proteins with the PHD domain ( Mellor, 2006 ), Pinskaya et al systematically tested yeast strains lacking different PHD proteins for an effect on GAL1–10 induction. They found that loss of Rco1, a component of the Rpd3S complex, mimicked the effects of set1 mutations on GAL1–10 expression. In addition, they used ChIP to show that Rpd3 is recruited to the repressed GAL1–10 locus and that this is abolished by H3K4A and set1 mutations. Interestingly, they did not observe any effect of a mutation deleting SET2, which encodes the H3K36 methyltransferase ( Lee and Shilatifard, 2007 ), on GAL1–10 induction kinetics, suggesting that the effects of GAL1ucut transcription might be mediated primarily through H3K4 methylation.

Although the different observations regarding the effects of GAL1ucut on induction kinetics and expression in low levels of glucose still need to be resolved, these papers indicate that cryptic transcription events might be used to set the chromatin-modification state of overlapping sequences. This regulatory strategy might be used more widely both groups present preliminary observations, suggesting that ncRNA might regulate expression of other yeast genes. Furthermore, in higher eukaryotes, ncRNA are implicated in genomic imprinting ( Edwards and Ferguson-Smith, 2007 ) and the function of some enhancers ( Drewell et al, 2002 ). Perhaps these transcription events serve to establish epigenetic marks that influence the function of the overlapping regulatory elements.


Functions and Mechanisms of Long ncRNAs

Like protein-coding genes, there is considerable variability in the function of long ncRNAs, yet clear themes in the data suggest that many long ncRNAs contribute to associated biologic processes. These processes typically relate to transcriptional regulation or mRNA processing, which is reminiscent of miRNAs and may indicate a similar sequence-based mechanism akin to miRNA binding to seed sequences on target mRNAs. However, unlike miRNAs, long ncRNAs show a wide spectrum of biologic contexts that show greater complexity to their functions.

Epigenetic Transcriptional Regulation

The most dominant function explored in lncRNA studies relates to epigenetic regulation of target genes. This typically results in transcriptional repression, and many lncRNAs were first characterized by their repressive functions, including ANRIL, HOTAIR, H19, KCNQ1OT1, and XIST (10, 47, 55). These lncRNAs achieve their repressive function by coupling with histone-modifying or chromatin-remodeling protein complexes.

The most common protein partners of lncRNAs are the PRC1 and PRC2 polycomb repressive complexes. These complexes transfer repressive posttranslational modifications to specific amino acid positions on histone tail proteins, thereby facilitating chromatin compaction and heterochromatin formation in order to enact repression of gene transcription. PRC1 may comprise numerous proteins, including BMI1, RING1, RING2, and Chromobox (CBX) proteins, which act as a multiprotein complex to ubiquitinate histone H2A at lysine 119 (61). PRC2 is classically composed of EED, SUZ12, and EZH2, the latter of which is a histone methyltransferase enzymatic subunit that trimethylates histone 3 lysine 27 (61). Both EZH2 and BMI1 are upregulated in numerous common solid tumors, leading to tumor progression and aggressiveness (13, 61).

Indeed, ANRIL, HOTAIR, H19, KCNQ1OT1, and XIST have all been linked to the PRC2 complex, and in all except H19, direct binding has been observed between PRC2 proteins and the ncRNA itself (40, 48, 55, 62, 63). Binding of lncRNAs to PRC2 proteins, however, is common and observed for ncRNAs, such as PCAT-1, which do not seem to function through a PRC2-mediated mechanism. It is estimated that nearly 20% of all lncRNAs may bind PRC2 (64), although the biologic meaning of this observation remains unclear. It is possible that PRC2 promiscuously binds lncRNAs in a nonspecific manner. However, if lncRNAs are functioning in a predominantly cis-regulatory mechanism—such as ANRIL, KCNQ1OT1, and XIST—then numerous lncRNAs may bind PRC2 to facilitate local gene expression control throughout the genome. Relatedly, studies of PRC2-ncRNA-binding properties have shown a putative PRC2-binding motif that includes a GC-rich double hairpin, indicating a structural basis for PRC2-ncRNA binding in many cases (40).

Similarly, PRC1 proteins, particularly CBX proteins, have been implicated in ncRNA-based biology. For example, ANRIL binds CBX7 in addition to PRC2 proteins, and this interaction with CBX7 recruits PRC1 to the INK4A/ARF locus to mediate transcriptional silencing (47). More broadly, work with mouse polycomb proteins showed that treatment with RNase abolished CBX7 binding to heterochromatin on a global level, supporting the notion that ncRNAs are critical for PRC1 genomic recruitment (65).

While PRC1 and PRC2 are perhaps the most notable partners of lncRNAs, numerous other epigenetic complexes are implicated in ncRNA-mediated gene regulation. For example, the 3′ domain of HOTAIR contains a binding site for the LSD1/CoREST, a histone deacetylase complex that facilitates gene repression by chromatin remodeling (Fig. 3A) (56). AIR is similarly reported to interact with G9a, an H3K9 histone methyltransferase (66). KCNQ1OT1 has been shown to interact with PRC2 (63), G9a (63), and DNMT1, which methylates CpG dinucleotides in the genome. More rarely, lncRNAs have been observed in the activation of epigenetic complexes. In a recent example, HOTTIP interacted with WDR5 to mediate recruitment of the MLL histone methyltransferase to the distal HoxA locus (52). MLL transfers methyl groups to H3K4me3, thereby generating open chromatin structures that promote gene transcription.

Mechanisms of lncRNA function. A, lncRNAs, such as HOTAIR, may serve as a scaffolding base for the coordination of epigenetic or histone-modifying complexes, including Polycomb repressive complexes and LSD1/CoREST. B, eRNAs transcribed from gene enhancers may facilitate hormone signaling by cooperating with lineage-specific complexes such as FOXA1 and AR. C, lncRNAs may directly affect tumor suppressor signaling either by transcriptional regulation of tumor suppressor genes through epigenetic silencing (e.g., ANRIL, top) or by mediating activation of tumor suppressor target genes (e.g., linc-p21, bottom). D, the MALAT1 lncRNA may be an integral component of the nuclear paraspeckle and may contribute to posttranscriptional processing of mRNAs. E, gene expression regulation may occur through direct lncRNA-mRNA interactions that arise from hybridization of homologous sequences and can serve as a signal for STAU1-mediated degradation of the mRNA. F, RNA molecules, including mRNAs, pseudogenes, and ncRNAs, can serve as molecular sponges for miRNAs. This generates an environment of competitive binding of miRNAs to achieve gene expression control based on the degree of miRNA binding to each transcript. The colored triangles represent different miRNA binding sites in a transcript. CDS, coding sequence.

In some cases, the mere act of lncRNA transcription is critical for the recruitment of protein complexes. Studies on H19, KCNQ1OT1, and AIR suggest that transcriptional elongation of these genes is an important component of their function (34, 53, 54). By contrast, other lncRNAs, including HOTTIP as well as many trans-regulatory lncRNAs, do not show this relationship (52). For these lncRNAs, biologic function may be centrally linked to their role as flexible scaffolds. In this model, lncRNAs serve as tethers that rope together multiple protein complexes through a loose arrangement. Supporting this model are the multiple lncRNAs found to bind multiple protein complexes, such as ANRIL (binding PRC1 and PRC2) and HOTAIR (binding PRC2 and LSD1/CoREST) (Fig. 3A).

Enhancer-Associated Long ncRNAs

In addition to facilitating epigenetic changes that impact gene transcription, emerging evidence suggests that some ncRNAs contribute to gene regulation by influencing the activity of gene enhancers. For example, HOTTIP is implicated in chromosomal looping of active enhancers to the distal HoxA locus (52), but knockdown and overexpression of HOTTIP is not sufficient to alter chromosomal confirmations (52). There is also a report of local enhancer-like ncRNAs that typically lack the H3K4me1 enhancer histone signature but possess H3K4me3 and function to potentiate neighbor gene transcription in a manner independent of sequence orientation (67).

A major recent development has been the discovery of eRNAs, which are critical for the proper coordination of enhancer genomic loci with gene expression regulation. Although the mechanism of their action is still unclear, in prostate cancer cells, induction of AR signaling increased eRNA synthesis at AR-regulated gene enhancers, suggesting that eRNAs facilitate active transcription on induction of a signaling pathway (29). Using chromatin conformation assays, Wang and colleagues (29) showed that eRNAs are also important for the establishment of enhancer-promoter genomic proximity by chromosomal looping. Moreover, eRNAs work in conjunction with cell lineage specific transcription factors, such as FOXA1 in prostate cells, thereby creating a highly specialized enhancer network to regulate transcription of genes in individual cell types (Fig. 3B) (29). Future work in this area will likely provide insight into signaling mechanisms important in cancer.

Modulating Tumor Suppressor Activity

The role of many lncRNAs as transcriptional repressors lends itself to inquiry as a mechanism for suppression of tumor suppressor genes. Here, one particular hot spot is the chromosome 9p21 locus, harboring the tumor suppressor genes CDKN2A and CDKN2B, which give rise to multiple unique isoforms, such as p14, p15, and p16, and function as inhibitors of oncogenic cyclin-dependent kinases. Expression of this region is affected by several repressive ncRNAs, such as ANRIL (Fig. 3C, top) and the p15-Antisense RNA, the latter of which also mediates heterochromatin formation through repressive histone modifications and has been observed in leukemias (47, 68).

Several lncRNAs are implicated in the regulation of p53 tumor suppressor signaling. MEG3, a maternally expressed imprinted lncRNA on Chr14q32, has been shown to activate p53 and facilitate p53 signaling, including the enhancement of p53 binding to target gene promoters (69). MEG3 has also been linked to p53 signaling in meningioma (70), and MEG3 overexpression suppresses cell proliferation in meningioma and hepatocellular carcinoma cell lines (70, 71). In human tumors, MEG3 downregulation is widely noted, with frequent hypermethylation of its promoter observed in pituitary tumors (10) and leukemias (72). Taken together, these data implicate MEG3 as a putative tumor suppressor.

A recently described murine lncRNA located near the p21 gene, termed linc-p21, has also emerged as a promising p53-pathway gene. In murine lung, sarcoma, and lymphoma tumors, linc-p21 expression is induced on activation of p53 signaling and represses p53 target genes through a physical interaction with hnRNP-K, a protein that binds the promoters of genes involved in p53 signaling (Fig. 3C, bottom) (73). linc-p21 is further required for proper apoptotic induction (73). These data highlight linc-p21 as a candidate tumor suppressor gene. However, due to sequence differences among species, it is currently unclear whether the human homolog of linc-p21 plays a similarly important role in human tumor development.

Regulation of mRNA Processing and Translation

While many lncRNAs operate by regulating gene transcription, posttranscriptional processing of mRNAs is also critical to gene expression. A primary actor in these processes is the nuclear paraspeckle, a subcellular compartment found in the interchromatin space within a nucleus and characterized by PSP1 protein granules (74). Although nuclear paraspeckle functions are not fully elucidated, this structure is known to be involved in a variety of posttranscriptional activities, including splicing and RNA editing (74). Paraspeckles are postulated to serve as storage sites for mRNA prior to its export to the cytoplasm for translation, and one study discovered a paraspeckle-retained, polyadenylated nuclear ncRNA, termed CTN-RNA, that is a counterpart to the protein-coding murine CAT2 (mCAT2) gene (75). CTN-RNA is longer than mCAT2, and under stress conditions, cleavage of CTN-RNA to the mCAT2 coding transcript resulted in increased mCAT2 protein (75).

In cancer, two ncRNAs involved in mRNA splicing and nuclear paraspeckle function, MALAT1 and NEAT1, are overexpressed. MALAT1 and NEAT1 are genomic neighbors on Chr11q13 and both are thought to contribute to gene expression by regulating mRNA splicing, editing, and export (Fig. 3D) (76, 77). MALAT1 may further serve as a precursor to a small 61-bp ncRNA that is generated by RNase P cleavage of the primary MALAT1 transcript and exported into the cytoplasm (78). Although a unique role for MALAT1 in cancer is not yet known, its overexpression in lung cancer predicts for aggressive, metastatic disease (79).

Regulatory RNA-RNA Interactions

Recent work on mechanisms of RNA regulation has highlighted a novel role for RNA-RNA interactions between ncRNAs and mRNA sequences. These interactions are conceptually akin to miRNA regulation of mRNAs, because sequence homology between the ncRNA and the mRNA is important to the regulatory process.

This sequence homology may be derived from ancestral repeat elements that contribute sequence to either the untranslated sequences of a protein-coding gene or, less frequently, the coding region itself. For example, STAU1-mediated mRNA decay involves the binding of STAU1, an RNA degradation protein, to protein-coding mRNAs that interact with lncRNAs containing ancestral Alu repeats. In this model, sequence repeats, typically Alus, in lncRNAs and mRNAs partially hybridize, forming double-stranded RNA complexes that then recruit STAU1 to implement RNA degradation (Fig. 3E) (80). A related concept is found with XIST, which contains a conserved repeat sequence, termed RepA, in its first exon. RepA is essential for XIST function, and the RepA sequence is necessary to recruit PRC2 proteins for X-chromosome inactivation (40).

Poliseno and colleagues (81) recently posited another model for mRNA regulation in which they suggested that transcribed pseudogenes serve as a decoy for miRNAs that target the protein-coding mRNA transcripts of their cognate genes. Sequestration of miRNAs by the pseudogene then regulates the gene expression level of the protein-coding mRNA indirectly (Fig. 3F). In addition to pseudogenes, this model more broadly suggests that all long ncRNAs, as well as other protein-coding mRNAs, may function as molecular “sponges” that bind and sequester miRNAs in order to control gene expression indirectly. These researchers showed that pseudogenes of two cancer genes, PTEN and KRAS, may be biologically active, and that PTENP1, a pseudogene of PTEN that competes for miRNA binding sites with PTEN, itself functions as a tumor suppressor in in vitro assays and may be genomically lost in cancer (81). This intriguing hypothesis may shed new light on the functions of ncRNAs, pseudogenes, and even the untranslated regions of a protein-coding gene.


Bombay Blood Group

The Hh Blood Group System

The Hh blood group system is composed of one gene (FUT1) located on chromosome 19q. It has four exons over 8 kbp of genomic DNA that encodes the gene product, 2-α-fucosyltransferase, an enzyme. The fucosyltransferase adds a fucose to a galactose located on type 2 carbohydrates on cellular surfaces. The function of the fucose may be in cellular adhesion or microorganism attachment. Homozygous genetic inactivation of the FUT1 or of the guanosine diphosphate (GDP)-fucose transporter gene (SLC35C1) can result in absent 2-α-fucoysltransferase enzyme activity, rendering red cell surfaces devoid of H antigen. Either genetic mechanism that leads to an H-deficient situation results in the Bombay (Oh). The Bombay phenotype usually results from a mutation in FUT1 combined with a nonsecretor phenotype (FUT2). The para-Bombay phenotype results from a mutation in FUT1 combined with a secretor phenotype (FUT2).

The phenotypic expression of H antigen is influenced by the ABO and secretor (FUT2) genes. The ABO gene product is a glycosyltransferase enzyme that adds an immunodominant carbohydrate to the H antigen the A phenotype is created by the 3-α-N-acetylgalactosaminyltransferase that adds GalNAc to the H antigen and the B phenotype is created by the 3-α-galactosaminyltransferase that adds galactose (Gal) to the H antigen the AB phenotype has both enzyme activities and the O phenotype (recessive) has no functional enzyme, usually due to a premature stop codon in the gene. The O phenotype has the highest H antigen density on the red cell surface (approximately 1.7 million per red cell) due to the absence of ABO glycosyltransferase activity. The frequency of the O phenotype is approximately 45% of Caucasians, 49% of Blacks, and 43% of Asians. The ABO glycosyltransferases cover up the H precursors proportionally to the relative enzymatic activity, although all cells (except for Oh and para-Bombay) carry some H antigen that remains unaltered by the ABO glycosyltransferases. The order from the highest number of H antigens on the red cell surface to the lowest is O > A2 > A2B > A1 > A1B. Because the ABO antigens are built on the H antigen precursor, they are not expressed in Bombay (Oh) phenotype when there are no H antigens present. The secretor (FUT2) gene encodes another fucosyltransferase that adds H antigen to secretions, such as saliva. People with para-Bombay phenotype have a mutated FUT2 (homozygous), rendering red cells devoid of H antigen, but have a functional FUT2 gene product (secretor) thus, H antigen is present in secretions and absent (or occasionally present in low levels) on red cells.

The H antigen occurs in 99.9% of all populations. The highest frequency of Oh phenotype is in people descended from the Indian subcontinent that is usually due to an inactivating missense mutation of FUT1 combined with the nonsecretor state. The prevalence in some regions of India is estimated to be 0.048% of the population. Bombay phenotype may be associated with consanguinity. Taiwan’s population also has a relatively high rate of Bombay phenotype (up to 0.012%).


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Imprinted Macro ncRNA Biology

We are in the early stages of understanding how the Airn and Kcnq1ot1 macro ncRNAs act to repress genes in cis. In particular, it is of interest to know if these two macro ncRNAs possess special properties relevant for their function that could also be typical of other macro ncRNAs. Both Airn and Kcnq1ot1 are RNAPII transcripts but are atypical RNPII transcripts because they are unusually large, mostly unspliced and not exported to the cytoplasm. 20 , 42 Both are described as ‘macro’ ncRNAs as Airn is 108 kb and Kcnq1ot1 is 91.5 kb ( Fig. 5 ). A small percentage (ρ%) of Airn transcripts are variably spliced to ncRNAs of approximately 1 kb and exported to the cytoplasm. Spliced Airn transcripts are 9 fold more stable than the unspliced transcript but are predicted to be non-functional. Kcnq1ot1 appears to lack any spliced variants ( Fig. 5 ). In contrast, the non-functional H19 ncRNA is a fully spliced ncRNA that is exported to the cytoplasm 43 however, it is atypical in that it has a low intron/exon ratio ( Fig. 5 ).

Genomic organization of imprinted macro ncRNAs. The genome position (UCSC Genome Browser on Mouse July 2007 Assembly), size and orientation of the 108 kb Airn ncRNA (A), the 91.5 kb Kcnq1ot1 ncRNA (B), and the 2.26 kb H19 ncRNA (C) is shown. The Airn and Kcnq1ot1 promoters are associated with CpG islands that lie in the ICE (white star), the H19 promoter lies 5 kb downstream of the ICE that is only moderately CG rich. Airn transcripts are mostly unspliced and nuclear-localized, but 5% of transcripts are spliced with the indicated organization and exported to the cytoplasm. 20 The Airn spliced variants (SV) all use the same splice donor (at +53 bp) but all use different 2 nd exons. The dotted rectangle indicates the full length of the unspliced Airn transcript as shown by oligo tiling array hybridization. 48 The small box inside the dotted rectangle indicates an annotated Airn EST. Kcnq1ot1 appears to lack spliced transcripts and is localized the nucleus. 42 H19 appears to be fully spliced but shows an unusually high exon/intron ratio and is exported to the cytoplasm. A biallelically-expressed and nuclear-localized transcript of unknown function has also been identified at the H19 ICE in embryonic liver tissue. 51 Note that Airn and Kcnq1ot1 have an antisense transcription overlap with one gene however, only Airn overlaps a promoter. The presence of interspersed repeats that are contained in the Airn and Kcnq1 but not H19 macro ncRNAs is shown underneath. H19 also contains the mir-675 miRNA. Airn contains a small oocyte RNA from the Au76 pseudogene (*).

While mammalian genes are often larger than 50 kb𠅎.g., the DMD gene is the largest known mouse gene that is 2,256 kb long and spliced to a 13.8 kb transcript containing 79 exons mature spliced mRNA transcripts are rarely larger than 10� kb. Thus mammalian mRNA genes normally have a high intron to exon ratio. Single exon mammalian mRNA genes do exist, however, they are normally short transcripts in the order of 1 kb that are exported to the cytoplasm. A consequence of the large size and unspliced nature of Airn and Kcnq1ot1 is that they contain transposons, which comprise approximately 50% of the mouse genome and are equally distributed in genes and intergenic regions. 44 Thus an additional atypical feature is that imprinted macro ncRNAs contain transposons, which are only rarely found in mRNAs ( Fig. 5 ). Since transposons are usually subject to DNA methylation and silenced in a differentiated cell genome, 45 mature RNAs containing transposon sequences are rare and open the possibility that they contribute to the repressor function of imprinted macro ncRNAs.

Experiments to identify functional regions within Airn and Kcnq1ot1 are in progress and we currently lack a clear picture of key regions within these ncRNAs. However, it is possible that the absence of splicing is related to macro ncRNA function. Exon-junction-complexes that mark spliced junctions may be used to target the nuclear export machinery and absence of splicing in macro ncRNAs may prevent nuclear export. The control of mRNA splicing is still a topic of active investigation. However, it is generally thought that RNAPII transcripts are co-transcriptionally processed to spliced products that are exported to the cytoplasm, because the elongating form of RNAPII recruits both splicing and polyadenylation proteins. 46 Recently, the polyadenylation complex was shown in yeast to recruit nuclear export factors. 47 Since RNAPII is recruited to the promoter, this may indicate a unique property of macro ncRNA promoters is to recruit a form of RNAPII that is unable to subsequently recruit splicing and polyadenylation factors. This has been directly tested for the Airn promoter by experiments that deleted the endogenous promoter and replaced it with the Pgk1 promoter that normally transcribes a 16.6 kb gene spliced to a 1.8 kb transcript containing 11 exons. 48 Surprisingly, the resultant Pgk-Airn transcript retained all properties of the endogenous Airn ncRNA. Pgk-Airn transcripts were 108 kb long and only produced a low level of spliced variants, and importantly, they retained the ability to repress Igf2r in cis. Thus the lack of Airn splicing appears to be regulated independently of its promoter sequence and RNAPII recruitment. This result contrasts with a study in yeast that swapped two mRNA promoters and showed that splicing regulation was promoter driven. 49 Further work will be needed to identify functional regions within the Airn and Kcnq1ot1 macro ncRNAs.


Methods

Data sources

In order to evaluate the performance of the approach, we prepared two kinds of data, ncRNA-target interaction matrix (LncRNADisease database (2015)) and target-disease interaction matrix (DisGeNET database) which are the same database sources used in Chen et al. (2013) [15], to form the tripartite network, as well as the sequence expression of those targets and ncRNAs (extracted from Uniprot and LncRNADisease databases, respectively). Since there are ncRNA sequence expressions and targets that are still unknown, we could only collect 76 ncRNAs, 109 targets, and 514 diseases (see Table 1). In Fig. 1, the degree distribution of the resulting network is shown. The results indicate that the entire network may follow a power law distribution. Moreover, we collected experimentally confirmed interactions between ncRNAs and miRNAs from the database shown in [16] and from Alaimo [13] Supplementary Information files and reconstructed another dataset composed on 151 ncNRAs, 179 targets and 134 diseases (see Helwak dataset in Table 1).


Watch the video: RNA interference RNAi: by Nature Video (May 2022).


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