Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.
We have investigated the roles of U1 and U2 snRNP particles in SV40 pre-mRNA splicing by oligonucleotide-targeted degradation of U1 or U2 snRNAs in Xenopus laevis oocytes. Microinjection of oligonucleotides complementary to regions of U1 or U2 RNAs either in the presence or absence of SV40 DNA resulted in specific cleavage of the corresponding snRNA. Unexpectedly, degradation of U1 or U2 snRNA was far more extensive when the oligonucleotide was injected without, or prior to, introduction of viral DNA. In either co-injected or pre-injected oocytes, these oligonucleotides caused a dramatic reduction in the accumulation of spliced SV40 mRNA expressed from the viral late region, and a commensurate increase in unspliced late RNA. When pre-injected, two different U2 specific oligonucleotides also inhibited the formation of both large and small tumor antigen spliced early mRNAs. However, even when, by pre-injection of a U1 5' end-specific oligonucleotide, greater than 95% degradation of the U1 snRNA 5' ends occurred in oocytes, no reduction in early pre-mRNA splicing was observed. In contrast, the same U1 5' end oligonucleotide, when added to HeLa splicing extracts, substantially inhibited the splicing of SV40 early pre-mRNA, indicating that U1 mRNP is not totally dispensable for early splicing. These findings confirm and extend our earlier observations which suggested that different pre-mRNAs vary in their requirements for snRNPs.
Pan
Z Q
ZQ
Department of Biological Sciences, Columbia University, New York, NY 10027.
Ge
H
H
Fu
X Y
XY
Manley
J L
JL
Prives
C
C
D000818:Animals; D001483:Base Sequence; D005260:Female; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008845:Microinjections; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear; D013539:Simian virus 40 / Q000235:genetics*; D014982:Xenopus laevis
D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear