Two Regions Promote U11 Small Nuclear Ribonucleoprotein Particle Binding to a Retroviral Splicing Inhibitor Element (Negative Regulator of Splicing)

Abstract

The Rous sarcoma virus (RSV) negative regulator of splicing (NRS) is an RNA element that represses splicing and promotes polyadenylation of viral RNA. The NRS acts as a pseudo 5 splice site (ss), and serine-arginine (SR) proteins, U1snRNP, and U6 small nuclear ribonucleoproteins (snRNPs) are implicated in its function. The NRS also efficiently binds U11 snRNP of the U12-dependent splicing pathway, which is interesting, because U11 binds only poorly to authentic substrates that lack a U12-type 3 splice site. It is of considerable interest to understand how the low abundance U11 snRNP binds the NRS so well. Here we show that U11 can bind the NRS as a mono-snRNP in vitro and that a G-rich element located downstream of the U11 site is required for efficient binding. Mutational analyses indicated that two of four G tracts in this region were important for optimal U11 binding and that the G-rich region did not function indirectly by promoting U1 snRNP binding to an overlapping site. Surprisingly, inactivation of U2 snRNP also decreased U11 binding to the NRS. The NRS harbors a branch point-like/pyrimidine tract sequence (BP/Py) just upstream of the U1/U11 site that is characteristic of 3 splice sites. Deletion of this region decreased U2 and U11 binding, and deletion of the G-rich region also reduced U2 binding. The G element, but not the BP/Py sequence, was also required for U11 binding to the NRS in vivo as assessed by minor class splicing from the NRS to a minor class 3ss from the P120 gene. These results indicate that efficient U11 binding to the isolated NRS involves at least two elements in addition to the U11 consensus sequence and may have implications for U11 binding to authentic splicing substrates.An important step in the production of most mRNAs is the removal of intron sequences from pre-mRNA by the process of RNA splicing. Different combinations of exons can be combined via alternative splicing to generate numerous proteins from a single gene (1), and it is now appreciated that regulated splicing accounts for great protein diversity (1-3). RNA splicing is also used by many viruses that infect eukaryotic cells as a means to expand their coding capacity and to regulate gene expression (4). This is true for retroviruses, with HIV 1 being an extreme example: it is estimated that more than 40 viral splice variants are produced in an HIV-infected cell (5-7). In addition to alternative splicing, retroviruses also exhibit incomplete splicing such that a large fraction of the primary transcripts remain unspliced and, in contrast to unspliced host cell mRNAs, are transported to the cytoplasm where they serve as mRNA and as genomes for progeny virions. These RNA processing events make retroviruses useful tools for studying the cellular RNA processing machinery, and determining how these events are controlled is required for understanding these important aspects of viral replication. RNA splicing takes place in a large macromolecular complex termed the spliceosome in which five small nuclear ribonucleoprotein partic...

Heterogeneous Nuclear Ribonucleoprotein H Is Required for Optimal U11 Small Nuclear Ribonucleoprotein Binding to a Retroviral RNA-processing Control Element

“…It was recently shown that a G-rich sequence downstream of the U11 consensus site is required for optimal U11 binding to the NRS in vitro and in vivo (30). Here, we show that the G-rich element specifically interacts with hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA-binding proteins (37).…”

“…For snRNP co-precipitation, Protein A-Sepharose beads were incubated with buffer, preimmune serum, or hnRNP H antibodies, and 10 l of beads were added to 50 l of nuclear extract under splicing conditions for 1 h. Samples were washed as above, treated with Proteinase K to release nucleic acids, and then phenol-extracted and subjected to Northern blotting with U1 and U11 riboprobes as described (30).…”

“…RNAs were incubated in complete or depleted HeLa nuclear extract under splicing conditions (47) with ATP for 30 min at 30°C and affinity-selected as described (30). Add-back assays utilized 200 ng of recombinant hnRNP H, which was the amount of hnRNP H found in a standard reaction.…”

“…is a low abundance minor class splicing factor and binds to the RSV NRS element remarkably well (29,30). It was recently shown that efficient U11 binding to the NRS in vitro and in vivo is dependent upon a G-rich region just downstream of the U11 consensus binding site (30).…”

“…An RNA-processing control element from Rous sarcoma virus (RSV), the negative regulator of splicing or NRS, harbors overlapping U1 and U11 binding sites and very efficiently binds U11 snRNP as well as U1 (29,30). This element represses viral splicing and splicing of heterologous introns in vitro and in vivo (29,(31)(32)(33), and it is required for efficient viral RNA polyadenylation (34,35).…”