U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells. Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation. To distinguish among these models and to test the function of U2 structural elements, we made >35 mutations in the yeast U2 snRNA gene. Some of the mutations were designed in pairs so that combinations could be made that would restore base-pairing to differentiate helix requirements from primary sequence requirements. The mutations identify an essential stem-and-loop structure adjacent to the branchpoint interaction region. A conserved complementarity to the loop just upstream of the Sm site and an additional conserved stem-loop are dispensable for U2 function, even in the background of a previously identified large internal deletion. Non-Watson-Crick base appositions at the 53-62 base pair in the essential stem lead to a variety of temperature and KCl-sensitive phenotypes, as well as an accumulation of unspliced precursors in vivo. Chemical structure probing of U2 RNA in vivo reveals that the bulk of U2 in a yeast cell adopts a structure in good agreement with that deduced from genetic results. We suggest that this stem-loop is not a binding site for an intrinsic U2 snRNP protein but may interact with other factors during spliceosome assembly or splicing.

Mutations in stem-loop Ila of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.RNA-RNA interactions between small nuclear RNAs (snRNAs) or between snRNAs and the pre-mRNA play critical roles in the accuracy and efficiency of splicing (for reviews, see references 15 and 16). Not all of these interactions are established simultaneously, nor do they persist once established. Rather, interactions are formed, modified, disrupted, and replaced during spliceosome assembly and splicing. The dynamic relationship between U4 and U6 has been known for some time (15, 16), and more recently it has been shown that U2 also interacts with U6 (11,26,47 Structure-function studies in this region of U2 show that only one of the structures, that containing stem-loop Ila, is absolutely essential for growth, arguing that the potential to form the others must be conserved for an accessory or overspecified function (2). Direct probing of yeast U2 structure in vivo confirms that the bulk of U2 in the cell adopts the essential structure (2); however, the alternative structure can form under certain circumstances, suggesting that this region of U2 snRNA is dynamic (49).An unanticipated phenotype associated with single base changes that destabilize the essential structure is cold-sensitive growth (2). Cold sensitivity is also observed in cell splicing extracts, allowing the demonstration of a role for stem Ila in the critical step of assembly of U2 snRNPs into the spliceosome in vitro (49). Structure probing experiments show that the bulk of U2 snRNA is misfolded in the cold-sensitive mutants, so that the RNA adopts the other phylogenetically conserved structure. Surprisingly, the misfolded form predominates at both permissive and restrict...

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs.[Keywords: Regulated splicing; regulons; splicing-sensitive microarray; epistasis] Supplemental material is available at http://www.genesdev.org.

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs. U2 small nuclear RNA (snRNA) is an essential component of the nuclear pre-mRNA splicing machinery (for reviews, see refs. 1 and 2) and is conserved in a wide range of organisms (3, 4). In spite of this, U2 function seems to differ in different species. For example, although an identical sequence near the 5' end of U2 pairs with the intron (5-7) during splicing in yeast and humans, U2-pre-mRNA binding requirements differ. In mammals, the polypyrimidine tract associated with the branchpoint region and ATP (1), as well as factor U2AF (8), are required. Other factors also influence polypyrimidine tract-dependent binding (8)(9)(10). In yeast, binding of U2 small nuclear ribonucleoprotein particles (snRNPs) to pre-mRNA is also ATP-dependent (11, 12); however, yeast introns do not always contain an obvious polypyrimidine tract (13), placing the need for factors analogous to mammalian U2AF in question. Instead, factors requiring the conserved yeast branchpoint sequence UACUAAC prior to U2 binding have been detected (11,14). Although mammalian U2 snRNPs have a branchpoint sequence-specific binding activity (15), a yeast intron is spliced in a human extract using sequences resembling a mammalian branchpoint, rather than at the more U2-complementary UACUAAC sequence (16).Dependence on assembly factors notwithstanding, the contributions of U2 structural variation and U2-intron pairing to U2 function are unclear. Mammalian splicing efficiency is influenced by but is not dependent on U2-branchpoint complementarity (6,7,(17)(18)(19) whereas in yeast this requirement seems more rigid (2). Precise selection of the branched nucleotide may also differ because in yeast, only the sixth position of the branchpoint sequence can form a branch (2), while in mammals multiple residues can be used (e.g., see ref. 20).Trypanosome mRNAs are matured by a trans-splicing process (21) that is U2-dependent (22). Trypanosoma brucei U2 is not conserved in the region of yeast and metazoan branchpoint interaction (3, 4). This may be peculiar to trypanosomes rather than to trans-splicing, because nematodes perfor...