The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.
We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.
Tarn
W Y
WY
Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan, Republic of China.
Lee
K R
KR
Cheng
S C
SC
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D053487:DEAD-box RNA Helicases; D000939:Epitopes; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005656:Fungal Proteins; C079877:PRP19 protein, S cerevisiae; D016760:Proto-Oncogene Proteins c-fos; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases