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Two Studies Uncover New Non-RNAi Roles for Argonaute-1, Dicer

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NEW YORK (GenomeWeb) — Two independent research teams this month published data revealing new roles for two key components of the canonical RNAi pathway — Argonaute-1 and Dicer — in the control of constitutive and alternate splicing, and in transcription termination and the maintenance of genomic stability, respectively.

The first report, which appeared in the Proceedings of the National Academy of Sciences, stems from the work of the University of Buenos Aires' Alberto Kornblihtt and collaborators who were investigating the roles of Argonaute (Ago) proteins in the mammalian cell nucleus.

Previously, his lab showed that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. This effect, they found, is dependent on Ago1 and involves a decrease in RNA polymerase II elongation.

Another group also recently described a similar effect over the variant region of the gene CD44, where both Ago1 and Ago2 were found to facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Recruitment of the Ago proteins, it was shown, required Dicer and the heterochromatin protein HP1.

Since then, two recent genome-wide surveys of Drosophila showed that Ago2 regulates alternative splicing as well as transcription of target genes, while studies in human cancer cells indicate that Ago1 interacts with RNA polymerase II and binds transcriptionally to active promoters, Kornblihtt and his team wrote in PNAS.

To elucidate the nuclear roles of Ago1 as they relate to alternative splicing in human cells, the scientists performed high-throughput DNA and RNA sequencing after Ago1 immunoprecipitation or Ago1 depletion.

Unexpectedly, they discovered that about 80 percent of Ago1 clusters are associated with cell type-specific transcriptional enhancers, most of which are overlapping active enhancers. Ago1 binding to active enhancers also appeared to be particularly associated with long small nuclear RNAs rather than short ones, and to be more prominent in intragenic, not intergenic, enhancers.

"Paradoxically, crossing ChIP-seq with RNA-seq data upon Ago1 depletion revealed that enhancer-bound Ago1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing," the investigators wrote in their paper.

Taken together, the findings provide evidence of an association of Ago1 to active transcriptional enhancers through the RNAs transcribed from them in combination with a control of alternative splicing, Kornblihtt and his team concluded.

In the second study, which ran in Cell, Robert Martienssen and colleagues at Cold Spring Harbor Laboratory (CSHL) were investigating the mechanisms underlying nuclear RNAi as a regulator of gene expression and epigenetic inheritance.

It has been shown that in the fission yeast S. pombi, RNAi is required to direct H3 lysine 9 dimethylation (H3K9me2) and H3K4 demethylation within the heterochromatic repeats flanking each centromere.

Further, it has been demonstrated that tightly regulated transcription within these repeats leads to the production of dsRNAs that are processed into siRNAs by the yeast's Dicer enzyme Dcr1. In such cases, small RNAs are loaded into Ago1, which guides it to complementary nascent RNA so that it can direct the deposition of H3K9me2 through the histone methyltransferase Clr4 via a process called cotranscriptional gene silencing.

Similar silencing mechanisms have been found in higher eukaryotes and had been thought to act on heterochromatic repetitive elements such as transposons, but recently small nuclear RNAs have been implicated in the regulation of RNA polymerase II at individual euchromatic genes, Martienssen and his colleagues wrote in Cell.

In fission yeast, this conserved function of RNAi in RNA polymerase II regulation is particularly important in the context of DNA replication. During S phase, which is the time when DNA replication occurs and epigenetic marks must be re-established, centromeric repeat units in the yeast are transcribed. This leads to a collision between RNA polymerase II and the replisome that is resolved by RNAi through the release of RNA polymerase II. In the absence of RNAi, however, homologous recombination leads to the restart of stalled replication forks, resulting in the loss of epigenetic modifications.

Using a genome-wide approach in S. pombe, the CSHL-led team found that Dcr1 coordinates transcription and replication outside of pericentromeres.

According to the paper, RNA polymerase II accumulation is a hallmark of polymerase collision, and Martienssen's lab previously found that RNA polymerase II accumulates in Dcr1-deficient cells at previously uncharacterized loci, including protein-coding genes, tDNA, and rDNA.

"Dcr1-dependent sRNAs were detected at these loci, but transcriptional termination was not dependent on sRNA biogenesis or other RNAi pathway components," demonstrating a Dcr1- specific role in RNA polymerase II release, the researchers wrote. Because these loci are strongly correlated with sites of replication pausing, they are likely to represent collisions between transcription and replication.

Focusing on one "particularly striking and unexpected" site of RNA polymerase II regulation, the subtelomeric rDNA repeats, the scientists found that Dcr1 is required for rDNA copy number maintenance.

The findings, the research group wrote, point to a novel genome-wide role for Dcr1 in S. pombe in terminating transcription by releasing RNA polymerase II at sites of collision between transcription and replication and thus maintaining genome stability.

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