Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis.

To further our understanding of the RNAi machinery within the human nucleus, we analyzed the chromatin and RNA binding of Argonaute 2 (AGO2) within human cancer cell lines. Our data indicated that AGO2 binds directly to nascent tRNA and 5S rRNA, and to the genomic loci from which these RNAs are transcribed, in a small RNA- and DICER-independent manner. AGO2 chromatin binding was not observed at non-TFIIIC-dependent RNA polymerase III (Pol III) genes or at extra-TFIIIC (ETC) sites, indicating that the interaction is specific for TFIIIC-dependent Pol III genes. A genome-wide analysis indicated that loss of AGO2 caused a global increase in mRNA expression level among genes that flank AGO2-bound tRNA genes. This effect was shown to be distinct from that of the disruption of DICER, DROSHA, or CTCF. We propose that AGO2 binding to tRNA genes has a novel and important regulatory role in human cells.

USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing.

Post-translational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of ubiquitin-specific peptidase 49 (USP49) as a histone H2B-specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons. USP49 forms a complex with RuvB-like1 (RVB1) and SUG1 and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression but affects the abundance of >9000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) levels at these exons as well as upstream 3' and downstream 5' intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B-specific deubiquitinase and uncovers a critical role for H2B deubiquitination in cotranscriptional pre-mRNA processing events.

Retroviral splicing suppressor sequesters a 3' splice site in a 50S aberrant splicing complex.

Retroviral replication requires both spliced and unspliced mRNAs. Splicing suppression of avian retroviral RNA depends in part upon a cis-acting element within the gag gene called the negative regulator of splicing (NRS). The NRS, linked to a downstream intron and exon (NRS-Ad3'), was not capable of splicing in vitro. However, a double-point mutation in the NRS pseudo-5' splice site sequence converted it into a functional 5' splice site. The wild-type (WT) NRS-Ad3' transcript assembled an approximately 50S spliceosome-like complex in vitro; its sedimentation rate was similar to that of a functional spliceosome formed on the mutant NRS-Ad3' RNA. The five major spliceosomal snRNPs were observed in both complexes by affinity selection. In addition, U11 snRNP was present only in the WT NRS-Ad3' complex. Addition of heparin to these complexes destabilized the WT NRS-Ad3' complex; it was incapable of forming a B complex on a native gel. Furthermore, the U5 snRNP protein, hPrp8, did not cross-link to the NRS pseudo-5' splice site, suggesting that the tri-snRNP complex was not properly associated with it. We propose that this aberrant, stalled spliceosome, containing U1, U2, and U11 snRNPs and a loosely associated tri-snRNP, sequesters the 3' splice site and prevents its interaction with the authentic 5' splice site upstream of the NRS.

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor.

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.

Packaging and reverse transcription of snRNAs by retroviruses may generate pseudogenes.

Retroviruses specifically package two copies of their RNA genome in each viral particle, along with some small cellular RNAs, including tRNAs and 7S L RNA. We show here that Rous sarcoma virus (RSV) also packages U6 snRNA at approximately one copy per virion. In addition, trace amounts of U1 and U2 snRNAs were detected in purified virus by Northern blotting. U6 snRNA comigrated with the RSV 70S genomic RNA dimer on sucrose gradients. We observed reverse transcription of U6 snRNA in an endogenous reaction in which RSV particles were the source of both reverse transcriptase and RNA substrates. This finding led us to examine mammalian genomic sequences for the presence of snRNA pseudogenes. A survey of the human, mouse, and rat genomes revealed a high number of spliceosomal snRNA pseudogenes. U6 pseudogenes were the most abundant, with approximately 200 copies in each genome. In the human genome, 67% of U6 snRNA pseudogenes, and a significant number of the other snRNA pseudogenes, were associated with LINE, SINE, or retroviral LTR repeat sequences. We propose that the packaging of snRNAs in retroviral particles leads to their reverse transcription in an infected cell and the integration of snRNA/viral recombinants into the host genome.