Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Abstract

Abstract. Temperature-sensitive mutations in the RNA2 through RNA/I genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.M UCH of the interest in expression of RNA polymerase lI transcribed genes is focused on defining the mechanism of processing the primary transcription products of these genes (pre-mRNAs) to cytoplasmic, translatable mRNAs. Accurate splicing of introns from pre-mRNAs is absolutely required for production of functional polypeptides from mosaic genes. The modulation of pre-mRNA processing patterns to generate polypeptide diversity during development of eukaryotic viruses (Nevins, 1982) and metazoan organisms (DeNoto et al., 1981; Schwarzbauer et al., 1983; Periasamy et al., 1985) is widespread, and elucidation of its molecular mechanisms is of great importance. This has become possible since the development of well-defined soluble in vitro systems that are capable of processing intron-containing exogenously added pre-mRNA molecules, using mammalian whole-ceil and nuclear extracts (Frendewey and Keller, 1985;Grabowski et al., 1985;Krainer and Maniatis, 1985) or yeast cell extracts (Brody and Abelson, 1985;Lin et al., 1985). Unique species with kinetic properties consistent with pre-mRNA processing intermediates have been identified in in vitro processing reactions Ruskin et al., 1984;Lin et al., 1985). The in vitro pre-mRNA processing mechanism appears to mimic reactions occurring in vivo, since similar intermediates have also been detected in mammalian cells (Wallace and Edmonds, 1983;Zeitlin and Efstratiadis, 1984) and yeast (Domdey et al., 1984;Rodriguez et al., 1984).Although the budding yeast Saccharomyces cerevisiae has a relatively small number of intron-containing genes (Gallwitz and Seidel, 1980;Ng and Abelson, 1980; Rosbash et al., 1981;Fried et al., 1981;Larkin and Woolford, 1983;Miller, 1984), the powerful molecular-genetic techniques available in this organism (Botstein and Davis, 1982), and the rapidly advancing cell biological technology make this an excellent system for studying pre-mRNA processing both in vivo and in vitro. The availability of a yeast in vitro splicing system (Brody and Abelson, 1985;Lin et al., 1985) provides the opportunity to analyze the functions of purified proteins in the pre-mRNA processing reaction.A unique advantage of studying pre-mRNA processing in yeast is the existence of 10 complementation groups of temperature-sensitive mutants, rna2 through rna// (Hartwell, 1967), that have been shown to be defective in premRNA processing (Rosba...

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

“…Perhaps RNAll protein is not easily accessible to the antibody, as suggested by our antibody inhibition experiments (Fig. 9) (32). Our immunofluorescence-determined nuclear location for wild-type levels of the RNA11 protein agrees with this study.…”

Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.

“…To date, the nature of the proteins encoded by sev eral of these temperature-sensitive mutants has been characterized. Using immunological tools, Last and Woolford (1986) have shown that the protein products of the RNA 2 and RNA 3 genes are nuclear proteins of -100,000 and 55,000 daltons, respectively. The RNA 8 gene specifies a 260,000-dalton protein that is complexed to U5 snRNA and, after incubation in the pres ence of ATP, also to U4 and U6 snRNAs (Lossky et al 1987).…”

PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle.

“…The PRP2 gene product is a 100-kDa protein (33) that functions in splicing after formation of the 40S spliceosome complex (35). PRP2, PRP3, and PRP11 have been localized in the nucleus (10,32). Genetic analysis has identified PRP16 (14) on the basis of suppression of a mutation in the branchpoint sequence of the precursor mRNA, which suggests that the PRP16 gene product may be part of the U2 snRNP or a U2 accessory factor.…”