Two regions promote U11 small nuclear ribonucleoprotein particle binding to a retroviral splicing inhibitor element (negative regulator of splicing).
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 3'ss 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.
McNally
Lisa M
LM
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Yee
Lily
L
McNally
Mark T
MT
D001120:Arginine / Q000737:chemistry; D001358:Avian Sarcoma Viruses / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D019921:Biotinylation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012342:RNA, Small Nuclear / Q000378:metabolism; D012190:Retroviridae / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection
D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C098762:U11 small nuclear ribonucleoprotein, human; C096986:U12 small nuclear RNA; D012694:Serine; D012313:RNA; D001120:Arginine