CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

Abstract

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,...

Promoter-driven splicing regulation in fission yeast

“…It is most similar to S. cerevisiae CUS2, which is associated with U2snRNA and is probably playing a role in the proper folding of this RNA (Yan et al, 1998). Uap2 is postulated to have a function mediating the U2AF-dependent association of the U2snRNP with the intron.…”

Dramatically reduced spliceosome in Cyanidioschyzon merolae

“…Another category of splicing factors that appear to be nearly absent in C. merolae is snRNP biogenesis proteins, the factors involved in initial assembly of snRNPs. The U2 protein Cus2, Aar2 from the cytoplasmic form of U5, Snu40 from the nuclear U5, Sad1 from the tri-snRNP, and the SMN protein required for correct Sm ring assembly in humans were not detected in C. merolae (59)(60)(61). SMN may be rendered unnecessary by the absence of stem-loops 3′ of the Sm binding site in C. merolae snRNAs.…”

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

“…5A). The non-essential CUS2 protein, which possesses an RNA recognition motif (RRM) and interacts genetically with U2 stem-loop IIa, could also participate in this U2 conformation change (38,60).…”

“…In addition, because the U2 snRNP-associated SF3 proteins interact with the anchoring site (18), we were also intrigued by the observation that SF3 proteins (59) can suppress mutations in U2 stem-loop IIa, suggesting that SF3 proteins and/or the U2 snRNP-associated CUS2 protein (60) could mediate a U2 conformational change between alternative phylogenetically conserved structures (49,(59)(60)(61)(62). Consistent with these earlier observations, we show here that (i) functional sequences upstream of the branch site are required for stable U2/BS association in complex A and for formation of U2/U6 helix II; and (ii) non-functional upstream sequences not only fail to support stable assembly of complex A, but cause misfolding of U2 stem-loop IIa.…”