We describe here a novel assay which allows the identification of factors required for the biosynthesis of spliceosomal snRNPs in yeast. Screening a collection of temperature-sensitive mutants resulted in the isolation of the sad1-1 mutant, a strain impaired in U4/U6 particle assembly.
While transcription and accumulation of the U4 snRNA appear to be unaffected, the U4 snRNA synthesized in sad1-1
cells at the nonpermissive temperature fails to assemble properly with the U6 snRNA and accumulates as free U4. A U4 particle is normally not detectable in wild-type cells, possibly due to the rapid kinetics of association of the newly synthesized U4 with the U6 snRNA, which is in excess (58
). In a sad1-1
background, the U4/U6 particle assembled before the temperature shift appears to be stable at the nonpermissive temperature, pointing to a requirement of Sad1p for U4/U6 biosynthesis, rather than stability of the particle or reassembly after each round of splicing. Deletion of BRR1
, which encodes a protein associated with all spliceosomal U snRNAs, results in degradation of newly synthesized U snRNAs (47
). Similarly, depletion of the core U snRNP protein Smd1p (52
), Smd3p (51
), or SmEp (5
) leads to destabilization of U snRNAs. In these mutants, failure to assemble the core U snRNP particle possible leads to degradation of U snRNAs. We have identified seven temperature-sensitive mutants which exhibit a similar phenotype: the levels of newly synthesized U4 are severely reduced at the nonpermissive temperature. In contrast, the accumulation of U4 snRNA in the sad1-1
mutant could be explained if Sad1p acted later in the biosynthetic pathway, when assembly of a core particle on U4 snRNA had already taken place, protecting the RNA moiety from degradation.
Sad1p appears to have a dual role in the cell. In addition to its involvement in the assembly of the U4/U6 particle, Sad1p is also required for splicing: sad1-1
cells accumulate endogenous pre-mRNAs at the nonpermissive temperature; the splicing of an intron-containing reporter gene is severely diminished in sad1-1
cells already at the permissive temperature, while it is abolished at the restrictive temperature; and additionally, extracts of sad1-1
cells fail to splice a pre-mRNA substrate in vitro. The accumulation of pre-mRNA, rather than splicing intermediates, indicates that Sad1p is required for the first step of splicing. We have so far been unable to immunodeplete Sad1p from extracts to a level that would inhibit splicing in vitro (data not shown). It is therefore formally possible that the splicing defect conferred by sad1-1
is indirect. It is, however, unlikely that the sad1-1
splicing defect is a consequence of the U4/U6 assembly defect (e.g., because of reduced levels of the U4/U6 particle), as it is evident early after the shift to the nonpermissive temperature, when there is still an abundant supply of wild-type U4/U6 and only a low level of free newly synthesized U4* (compare Fig. , lanes 2, to Fig. B). On the other hand, the U4/U6 assembly defect does not appear to be a nonspecific secondary effect of the defective splicing of sad1-1
mutants (e.g., due to the depletion of an intron-containing assembly factor), as the majority of the splicing mutants tested have no U4/U6 assembly defect. A dual requirement for U snRNA biosynthesis and splicing is expected for U snRNP protein components. For instance, the U snRNA-associated Brr1p, Smd1p, Smd3p, and SmE (5
) are required for splicing as well as snRNP biosynthesis. However, we could not detect an association of Sad1p with any of the spliceosomal U snRNAs under different salt conditions, making it unlikely that Sad1p is a stably associated snRNP protein.
Sad1p is essential for cell viability, and it localizes to the nucleus at steady state, consistent with an involvement in splicing and in a late step in snRNP assembly. Sequence analysis reveals, in addition to a nuclear localization signal, the presence of a putative zinc finger motif of the C2
type at the N terminus of the protein, similar to the one present in the splicing factors Prp6p, Prp9p, Prp11p, and the U1C protein (29
). The role of this motif in spliceosomal proteins is unknown, but it is speculated to mediate interactions with U snRNAs or the mRNA substrate or to be involved in protein-protein interactions. Homologues of Sad1p were identified in Arabidopsis
, C. elegans
, and human, all of which contain the C2
Thirteen splicing-defective prp strains were tested for their ability to assemble the U4/U6 particle at the nonpermissive temperature. While the majority of the splicing mutants tested had no U snRNP assembly defects, two groups of mutants exhibited a SAD phenotype.
Prp3p, Prp4p, and Prp24p are associated with the U6 and U4/U6 particles and have been proposed to be involved in promoting dissociation-reassociation of U4 with U6 snRNAs during the spliceosomal cycle (1
). Consistent with their proposed role, we show that the vast majority of U4 snRNA present in prp3-1
, or prp24-1
cells at the nonpermissive temperature was not associated with U6. While at the permissive temperature, assembly of the U4/U6 particle took place (albeit inefficiently, especially for prp24-1
cells), soon after a shift to the nonpermissive temperature, both the U4 snRNA present from before the temperature shift and newly synthesized U4 snRNA accumulated as free U4. The levels of U6 snRNA were severely reduced at the nonpermissive temperature (4
), while free U4 snRNA was stable.
Strains harboring the prp17-1
) are also defective in the assembly of the U4/U6 snRNP, but, in contrast to prp3-1
, and prp24-1
strains, they accumulate both free U4 and U6 snRNAs. Prp17p is required for the second step of splicing and genetically interacts with the U5 snRNP (15
). Prp19p, which is not tightly associated with any U snRNA (63
), becomes associated with the spliceosome concomitantly with or just after dissociation of the U4 snRNA (63
) and is present in extracts in a complex with a number of unidentified proteins (62
). The prp17-1
mutation could be blocking the reassociation of U4/U6 following splicing. While mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p probably destabilize the particle and expose U6 snRNA to degradation, mutations in Prp17p and Prp19p might block the spliceosome at a stage where U4 has dissociated from U6.
sad1-1 appears to be unique among the splicing-defective mutations tested in affecting the assembly of newly synthesized U4 into the U4/U6 particle rather than the stability of the assembled U4/U6 snRNP or the reassociation of U4 and U6 following each round of splicing. The involvement of Sad1p in U4/U6 assembly could be independent from its function in splicing. Alternatively, the block imposed in splicing in the sad1-1 mutant could result in titrating a factor that is rate limiting for U4/U6 assembly. Elucidation of the role of Sad1 has to await a more detailed analysis of its biochemical function and would be aided by the characterization of its higher eukaryotic homologues and their role in snRNP assembly in vertebrates. Additionally, it is hoped that characterization of the remaining mutants identified by the screen will shed more light on the U snRNP assembly pathway in yeast.