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
). 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 cross-linking 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
). 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 pre-mRNA 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 5′ splice site by the U1 snRNP and at the branch point by the homologous mammalian (SF1) or yeast (BBP) branch point-interacting proteins (2
). Formation of this complex is expected to specify an exon joining event because the 5′ splice site and branch point are selected, and in most cases the 3′ splice site used will be the first YAG downstream from the branch point (54
). The ATP-dependent binding of U2 snRNP to the complex presumably seals its fate. This reaction forms the prespliceosome (called the A complex in mammals and the B complex or III in yeast) and the subsequent steps of spliceosome assembly and splicing proceed.
Prior to its stable docking with the commitment complex to form the prespliceosome, an assembly-competent U2 snRNP must be formed. A number of requirements for U2 participation in prespliceosome formation have been identified through both yeast genetics and biochemical approaches in the mammalian system. In yeast, stem-loop IIa of U2 RNA contributes to this binding through a network of functional interactions with the products of the PRP5
, and CUS1
). By several criteria, the PRP9, PRP11, and PRP21 proteins are homologs of the subunits of mammalian SF3a, also called SAP61, -62, and -114 or SF3a60, -66, and -120, respectively (for reviews, see references 32
). The CUS1 and HSH49 proteins are yeast splicing factors homologous to two mammalian SF3b subunits, SAP145 and SAP49 (21
). SF3a and SF3b are distinct multimeric protein complexes that bind to the 12S form of the mammalian U2 snRNP to create the 17S form that is recruited to the spliceosome (for a review, see reference 39
). SF3a binding to U2 is dependent on binding of SF3b (18
), and additional proteins are found associated with the 17S U2 snRNP (10
). Once assembled into the spliceosome, this set of proteins and U2 RNA remain through both steps of splicing (11
Despite the enumeration of these components, none have been shown to interact directly with U2 RNA and little is known about how their assembly with the core U2 snRNP is regulated. In this report, we describe CUS2
, a novel gene whose mutation improves the performance of a cold-sensitive stem IIa mutant U2 RNA (5
). The CUS2 protein is an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins (16
) closely related to human Tat-SF1, identified as a host cofactor important for Tat-dependent, transactivating region (TAR)-dependent human immunodeficiency virus (HIV) transcription (72
). We show that Tat-SF1 and CUS2 have parallel associations with the splicing protein homologs SAP62 and PRP11. The data provide evidence that CUS2, and by inference Tat-SF1, is a splicing factor that aids assembly of the splicing-competent U2 snRNP in vivo.