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

Abstract

The yeast rna mutations (rna2 through rnalOIII) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rnall extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNAll-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNAll-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA1 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.Through the development of in vitro splicing systems, a two-step biochemical pathway for the removal of introns from nuclear mRNA precursors (pre-mRNAs), referred to as mRNA splicing, has been delineated (for a review, see reference 42). This splicing reaction is done on a 40S to 60S macromolecular complex, the spliceosome, on which the reaction intermediates are confined (4,15,16). It is becoming apparent that the complexity of such a large particle may well be as great as that of the ribosomal subunits and that it could contain a number of proteins and several RNAs.To date, five major classes of small nuclear RNAs (snRNAs; Ul, U2, U4 to U6) have been found to be involved in mammalian mRNA splicing (for a recent review, see reference 37). Of these, four (Ul, U2, U4, and U5) possess a 5' trimethylguanosine (m3G) cap. All five snRNAs are tightly associated with one or more proteins and function in the form of small nuclear ribonucleoprotein particles (snRNPs) (48, 56). The human autoimmune antiserum antiSm has been used to precipitate these snRNAs and to demonstrate that these snRNAs are required for mRNA splicing (28, 43). The Sm antigens are a subset of snRNP proteins found in all five of the snRNPs which function in mRNA splicing (48). In yeasts, snRNAs also possess the 5' m3G cap structure which renders them immunoprecipitable by the anti-m3G antibody (reviewed in reference 18). Furthermore, a subset of yeast snRNAs (e.g., snR7, snR14, and snR20) contain potential Sm antigen-binding sites in their single-stranded regions with a consensus sequence of A(U3-)G (2,19,44,49,50,60 for mammalian cell systems; snR6, snR7, snR14, and snR20 for the yeast system) reside on the spliceosomes (8,17,26,27,46,47). In addition, the involvement of the heterogeneous nuclear RNP (hnRNP) complexes in mRNA splicing and spliceosome formation has been implicated by inhibition experiments with antibodies against the hnRNP core proteins (9, 53). Spliceo...

The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition.

“…6). One of these temperature-sensitive mutants, prpll (Chang et al 1988), normally grows well at 25°C but failed to grow at this temperature in combination with the MUD2 KO gene. The synthetic lethality was not attributable to some general difficulty of MUD2KO-prp mutant combinations: The growth of two other prp temperature-sensitive mutants, another U2 snRNP temperature-sensitive mutation (prp9, Abovich et al 1990) and a U5 snRNP temperaturesensitive mutation (prp8, Lossky et al 1987) was unaffected by the addition of the MUD2 KO allele.…”

“…Primers were designed from the PRP11 sequence (Chang et al 1988). This amplified DNA was cloned in the above-described plasmid in place of the BarnHI-SalI fragment coding for MUD2 HA.…”

The biochemical defects ofprp4-1andprp6-1yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP

“…However, genetic approaches have led to the isolation of many splicing deficient mutants, called prp mutants (20,21). Some PRP genes encode snRNP components, such as PRP4, PRP8 and PRP24 (14,19,(22)(23)(24) and others, such as PRP2, PRP5, PRP9, PRP11, PRP16, PRP22 and PRP28, are implicated in spliceosome assembly or disassembly (25)(26)(27)(28)(29)(30)(31).…”

Homodimerization of the human U1 snRNP-specific protein C

“…The N-terminal region of U1C, containing a zinc finger-like sequence which resembles the CC-HH zinc fingers of the TFIIIa type, is required and sufficient for this interaction (10). Similar CH motifs have been observed in the yeast splicing proteins PRP6, PRP9 and PRP1 1 (11,12). Neither one of the proteins U1C, PRP6, PRP9 or PRP1 1, however, has actually been shown to bind zinc.…”