A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions.

Abstract

The Neurospora crassa cyt4 mutants have pleiotropic defects in mitochondrial RNA splicing, 5' and 3' end processing, and RNA turnover. Here, we show that the cyt-4 gene encodes a 120-kDa protein with significant similarity to the SSD1/SRK1 protein of Saceharomyces cerevisiae and the DIS3 protein of Sclhizosaccharomyces pombe, which have been implicated in protein phosphatase functions that regulate cell cycle and mitotic chromosome segregation. The CYT-4 protein is present in mitochondria and is truncated or deficient in two cyt4 mutants. Assuming that the CYT-4 protein functions in a manner similar to the SSD1/SRK1 and DIS3 proteins, we infer that the mitochondrial RNA splicing and processing reactions defective in the cyt4 mutants are regulated by protein phosphorylation and that the defects in the cyt4 mutants result from failure to normally regulate this process. Our results provide evidence that RNA splicing and processing reactions may be regulated by protein phosphorylation.The processing and splicing of mitochondrial RNAs play a key role in expression of mitochondrial genes in Neurospora crassa and other organisms. In N. crassa, mutants defective in splicing the group I intron in the mitochondrial large rRNA have been identified among strains deficient in cytochromes b and aa3. In an initial screen, we identified eight such mutations, which mapped to three nuclear genes, cyt-18, cyt-J9, and cyt4 (1, 2). More recently, we found an additional mutant in the cyt4 gene, which others had mapped erroneously to the cyt-) gene (A. J. Snook and A.M.L., unpublished data). The mutations in the cyt-18, cyt-19, and cyt4 genes are recessive to their wild-type alleles and complement each other in heterokaryons (2). Thus, the genetic analysis indicates that at least three trans-acting polypeptides are required for splicing the group I intron in the mitochondrial large rRNA in vivo.Of the genes involved in mitochondrial RNA splicing, the one studied in the greatest detail has been the cyt-18 gene, which encodes a mitochondrial tyrosyl-tRNA synthetase that functions in both splicing and aminoacylation (3). Temperature-sensitive mutants in the cyt-18 gene are defective in mitochondrial protein synthesis and in splicing the mitochondrial large rRNA intron and other group I introns in mitochondrial mRNAs (1, 4). The mitochondrial tyrosyl-tRNA synthetase binds directly to the intron RNA and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro (5, 6). The cyt-19-1 mutant is defective in splicing the same group I introns as the cyt-18 mutants and is presumed to be defective in a second component that functions directly in splicing, perhaps by contributing to the correct folding of the precursor RNAs or by facilitating binding of the CYT-18 protein (7,8). The mutations in the cyt-18 and cyt-19 genes specifically affect the splicing of group I introns and have relatively little effect on other mitochondrial RNA processing reactions (1, 4, 7).By contrast, the cyt4 mutants have a complex phenotype w...

Nucleocytoplasmic shuttling of Ssd1 defines the destiny of its bound mRNAs

“…Ssd1 is the first example of a yeast mRNP that shuttles into and out of the nucleus and has a role both in polarized mRNA localization and in translational repression. This complexity of Ssd1 function coupled with the diversity of its associated mRNAs likely account for the large variety of SSD1 genetic interactions (Sutton et al, 1991;Chiannilkulchai et al, 1992;Turcq et al, 1992;Stettler et al, 1993;Uesono et al, 1994;Tsuchiya et al, 1996;Luukkonen and Seraphin, 1999;Moriya and Isono, 1999;Kaeberlein and Guarente, 2002;Wheeler et al, 2003;Kaeberlein et al, 2004;Tanaka et al, 2007;Mir et al, 2009).…”

Ssd1p of Saccharomyces cerevisiae Associates with RNA

“…II-related Proteins-It has been reported that the Ssd1 protein shows weak similarity with the Cyt4 protein, which is related to a mitochondrial RNA splicing and processing factor of N. crassa (16). Another report demonstrated the conserved domains in several proteins involving not only Cyt4p but also RNase II, a 3Ј to 5Ј exoribonuclease, encoded by rnb of E. coli (14).…”

“…In recent years, it has been reported that SSD1 has a weak but significant similarity with dis3 ϩ of Schizosaccharomyces pombe (6,13), DSS1 of S. cerevisiae (14), vacB of Shigella flexneri (15), cyt4 of Neurospora crassa (16), zam of Synechocytosis PCC 6803 (17), and rnb of Escherichia coli (18). Some of these genes are known, or implied, to be involved in the modification of RNAs: 1) cyt4 is required for the mitochondrial rRNA splicing and processing reaction; 2) DSS1 is a multicopy suppressor of the disruptant of SUV3 encoding a putative RNA helicase-like protein; 3) the vacB mutation reduces the level of the virulence antigens, IpaB, IpaC, IpaD, and VirG, at the post-transcriptional level; and 4) the RNase II encoded by rnb has a 3Ј-to-5Ј exoribonuclease activity.…”

A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803

“…The VacB protein from Shigella flexneri is involved in the control of virulence of this pathogenic bacterium, but its mode of action is unknown [26]. A highly conserved domain covering about 30 amino acids (residues 548 to 577 of Zam) is found in all three, and also in the CYT4 protein from Neurospora crassa, involved in splicing of mitochondrial RNAs [27]. …”