A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.In eukaryotes, the removal of introns from nuclear transcripts requires two transesterification reactions carried out by spliceosomes. Five small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U5, U4, and U6, and many extrinsic protein factors act in concert to build a spliceosome and execute the splicing reactions (38,39,49,50,53). The spliceosome is clearly the most dynamic of the RNP enzymes. Major changes in the secondary structure of U4, U6, and U2 snRNAs during the spliceosome cycle are inferred from genetic and crosslinking studies (reviewed in reference 63). The snRNAs arrive at the assembling spliceosome in a form unlike that necessary for the catalytic activity of splicing and must be rearranged before splicing can proceed (7,45). A less studied corollary of this finding is that these rearrangements must be undone during spliceosome disassembly, and snRNA structure must be regenerated in an appropriate form for another round of spliceosome assembly and splicing (56). Although individual proteins are clearly linked to changes in the composition and organization of splicing complexes at distinct points in the splicing pathway (for a review, see reference 63), it has been difficult to assign responsibility for a specific RNA rearrangement event to any single protein.The first ATP-dependent step during in vitro spliceosome assembly is the stable binding of the U2 snRNP to the branch point region of the intron, an event normally dependent on formation of ATP-independent complexes between the premRNA and other proteins, as well as the U1 snRNP (51). These ATP-independent complexes (called the E complex in mammalian studies and commitment complexes in yeast studies) contain pre-mRNA that has been recognized both at the 5Ј splice site by the U1 snRNP and at the branch point by the homologous mammalian (SF1) or yeast (BBP) branch pointinteracting proteins (2,13,29,38,48). Formation of this complex is expected to specify an exon joining event because the 5Ј splice site and branch point are selected,...

U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 stem I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA.Nuclear pre-mRNA splicing is a dynamic process marked by an intricate program of RNA-RNA, RNA-protein, and protein-protein interactions (for reviews, see references 6, 43, and 47-49). Five small nuclear RNAs (snRNAs) (U1, U2, U4, U5, and U6), packaged with proteins into small nuclear ribonucleoprotein particles (snRNPs), are essential components of spliceosome. The snRNAs, especially U2 and U6, are intimately associated with the reactive regions of the intron during the chemical reactions of splicing (6,43,(47)(48)(49), supporting the hypothesis that the splicing reactions are RNA catalyzed (14,55). Construction of the catalytically active spliceosome is heavily dependent on the activity of numerous protein factors, including snRNP proteins (29,47,48). Thus, snRNA sequences contain information necessary for interaction with the substrate, each other, and the proteins that guide them to their places in the activated spliceosome.A web of RNA-RNA interactions holds U2, U6, the 5Ј splice site, and the branchpoint together for the first step of splicing (for reviews, see references 6, 43, 48, and 49). In both Saccharomyces cerevisiae and mammals, the GUAGUA sequence of U2 snRNA base pairs with the intron branchpoint (51, 66, 71). Two other stretches of U2 RNA are involved in forming helices with U6 snRNA. The 5Ј end of U2 snRNA base pairs with the 3Ј end of U6 snRNA to form U2-U6 helix II (Fig. 1) (28), a structure that is important for splicing in mammalian cells (18, 67) but can be changed without greatly compromising splicing...

Active surveillance is a management strategy for many low-grade prostate cancers. Repeat biopsies monitor for previously undetected high-grade cancer. We show that a model with clinical variables, including a panel of four kallikreins, indicates the presence of high-grade cancer before a biopsy is performed.

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