Ssd1p of Saccharomyces cerevisiae Associates with RNA

Abstract

The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4, slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm.Cellular RNAs do not exist as a free form but as an RNAprotein complex. The proteins that directly associate with RNA are thought to play important roles in the regulation of gene expression at the post-transcriptional level (1, 2). In eukaryotic cells, proteins that bind to RNA polymerase II transcripts include both heterogeneous nuclear RNA-binding proteins and cytoplasmic mRNA-binding proteins. Heterogeneous nuclear RNA-binding proteins bind pre-mRNAs and are associated with them during the processing events required for the formation of mature mRNA (1). Once mRNAs are transported to the cytoplasm, they form cytoplasmic mRNA-binding protein complexes (2). Cytoplasmic mRNA-binding proteins seem to regulate translation, localization, or stability of mRNA (3). At present, many RNA-binding proteins have been isolated and characterized (4, 5), but their functions have not been fully understood.In Saccharomyces cerevisiae, the SSD1 gene has been first characterized to suppress the sit4 mutation defective in a protein phosphatase subunit (6). Not only in this case, but also in many other cases, SSD1 has been isolated as a single copy suppressor of mutation defective in RPC31 encoding a subunit of RNA polymerase III (7), in PDE2 encoding the cyclic AMP phosphodiesterase (8), in BCK1 encoding mitogen-activated protein kinase kinase kinase (9), in MPK1 encoding mitogenactivated protein kinase (10), or in G 1 cyclin (11). These reports indicate that SSD1 is involved in many systems. Sutton et al.also reported that there are two alleles of the SSD1 gene; one is called ssd1-d (dead) and the other is called SSD1-V (viable). They described that SSD1-V could suppress the double mutations of ssd1-d and sit4 (6). We have also isolated the SSD1 gene as the MCS1 gene involved in stable maintenance of the minichromosome (12). The SSD1/MCS1 gene product was detected as a ϳ160-kDa protein in certain wild type strains bearing SSD1-V, such as KA31 or RAY-3A, whereas a protein of this size was not detected in another wild type strain bearing ssd1-d, such as YPH499 (7). These findings indicate that SSD1-V is simply a wild type gene and ssd1-d is a ...

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…It is also conceivable that Ssd1 performs additional functions in the cell independent of its phosphoCTD association. This would be consistent with the finding that Ssd1 can be found both in the nucleus and in the cytoplasm (49). Functions of Ssd1 independent of the phosphoCTD made by CTDK-I would also help to explain phenotypes displayed by various ssd1 and ctk1 mutants.…”

“…Ssd1, a member of the ribonuclease II family of proteins ( Figure 4A), has been implicated in diverse pathways regulating cell growth and differentiation (e.g., refs [46][47][48][49][50][51][52][53][54][55][56]. It has been shown to interact genetically with many different proteins, but very little is known about its biochemistry.…”

“…It has been shown to interact genetically with many different proteins, but very little is known about its biochemistry. Despite its being in the RNase II family, no RNase activity has been demonstrated for Ssd1; however, it can bind RNA and is thought to be RNA-associated in vivo (49). SSD1 is polymorphic, and four different alleles having been identified in laboratory strains: SSD1-υ1, SSD1-υ2, ssd1-d1, and ssd1-d2.…”

Involvement of Nst1p/YNL091w and Msl1p, a U2B″ splicing factor, in Saccharomyces cerevisiae salt tolerance

“…The fact that MSL1 encodes a splicing factor and that Msl1p function is required for the effect of NST1 on salt tolerance stresses the importance of efficient RNA processing under salt stress conditions. Interestingly, the SSD1 gene, which affects lithium tolerance (see above) without significantly altering intracellular lithium accumulation (data not shown), also is able to associate with RNA (Uesono et al, 1997) and was identified as a general splicing mutant suppressor (Luukkonen and Seraphin, 1999). Finally, a novel yeast genomewide two-hybrid screen has revealed protein-protein interactions between the NST1/YNL091w gene product and Lsm4p, a Sm-like protein associated with spliceosome assembly (Fromont-Racine et al, 2000).…”

Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation inSaccharomyces cerevisiae

“…The insertion sites were located at 1317 base pairs and 2164 base pairs downstream of the start codon of the 3753-base pair open reading frame of SSD1. Because the C-terminal conserved region of Ssd1p is required for its function (Uesono et al, 1997), both transposon insertions were likely to result in loss-of-function alleles of SSD1. SSD1 is known to genetically interact with many different genes but its physiological function is not clear.…”