Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.
The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo.
The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in $accharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prpll and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.[Key Words: U2 snRNA; Saccharomyces cerevisiae; spliceosome assembly; pre-mRNA] Received August 25, 1995; revised version accepted October 24, 1995.The spliceosome is the large ribonucleoprotein particle responsible for removal of introns from nuclear premRNA transcripts. Spliceosome assembly and function are complex and dynamic, involving multiple transient protein-protein, protein-RNA and RNA-RNA interactions. The spliceosome is built on an intron-containing transcript by the sequential recognition of conserved sequence elements near the reactive sites. A transcript first becomes committed to the splicing pathway by stable association with the U1 small nuclear ribonucleoprotein (snRNP) to form the commitment complex, a step that does not require ATP. Stable binding of U2 snRNP to the commitment complex near the pre-mRNA branchpoint requires ATP and forms the prespliceosome. With the binding of US/U4.U6 tri-snRNP the spliceosome is assembled but must be activated before the cleavage and ligation reactions begin (for review, see Moore et al. 1993;Newman 1994;Nilsen 1994; Ares and Weiser 1995}.The recognition of an intron and choice of splice sites are likely accomplished during the early steps of spliceosome assembly and may hold the key to the regulation of splicing. As the first ATP-requiring step, the stable addition of the U2 snRNP at the branchpoint is an attractive potential site of regulation. The RNA moiety of the U2 snRNP plays two roles in this step. A region called the branchpoint interaction sequence forms base pairs with the pre-mRNA at the site of lariat formation {Parker et Wu and Manley 1989; Zhuang and Weiner 19891. A structure downstream of the branchpoint interaction sequence called stem-loop...
Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10prp mutations and found lethal double-mutant combinations withprpS, -9, -11, and -21 but not with prp3, 4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prpllp, and Prp2lp) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -llp, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop Ila and mediate the association of the U2 snRNP with pre-mRNA.Splicing of nuclear pre-mRNA requires a sophisticated ribonucleoprotein (RNP) complex called the spliceosome. The spliceosome is built on an intron-containing transcript by the sequential binding of small nuclear RNPs (snRNPs) to each other and to specific sites on the transcript, so that the pre-mRNA is properly arranged for splicing (20,21). Before, during, and after the cleavage-ligation steps of splicing take place, the spliceosome is acted upon by a series of extrinsic factors, some of which bind transiently and trip a limited set of steps in the sequence (15,29,48). The events that define the transition from one state to the next during spliceosome assembly and function are both compositional and conformational: snRNPs and extrinsic factors are added and removed, and RNA-RNA interactions between the small nuclear RNAs (snRNAs) or between the snRNAs and the pre-mRNA within the spliceosome may be established and dissolved (42). Splicing factors include a family of proteins with ATP binding domains suggesting a means for driving the process forward and ensuring the accuracy of events (20,21).The expectation that alternative splicing is achieved by regulated spliceosome assembly has placed the early steps of splicing complex formation under close scrutiny. Using substrate commitment experiments, several investigations have led to a similar set of conclusions for both systems (27,32,38,49,50
Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.
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