The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00438-006-0161-5 and is accessible for authorized users

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexe

C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis.

In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3\u27 phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3\u27 phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2

The transcription machinery in schizosaccharomyces pombe and its regulation

The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the general transcription machinery. The Mediator was originally identified in Saccharomyces cerevisiae and is required for the basal and regulated expression of nearly all RNA polymerase II dependent genes. Mediator-like complexes have also been identified in higher eukaryotes and shown to play an essential role in transcription regulation. However, most of the subunits identified in these mammalian complexes displayed low or no significant sequence similarity with Mediator subunits previously identified in yeast. Our specific aim was to purify Mediator from Schizosaccharomyces pombe and to compare its subunit composition and function to S. cerevisiae and mammalian Mediators to shed light on the mechanism and evolution of Mediator dependent transcription regulation. In paper I and II, we purified the S. pombe Mediator in complex with RNA polymerase II. We showed that the S. pombe Mediator complex was considerably smaller than its S. cerevisiae counterpart containing only 13 subunits instead of 20. Three of the S. pombe subunits were species specific named PMC for Pombe Mediator Complex. Additionally, the S. pombe Mediator contained 10 subunits conserved in S. cerevisiae and 8 in metazoans. Genetics showed that the conserved subunits were essential for cell growth, whereas the species-specific subunits were non-essential. Our findings led us to propose that the Mediator consists of a set of core subunits conserved through evolution that is responsible for contacts with the general transcription machinery and a set of species-specific subunits that function as a dynamic interface for direct interactions with gene-specific activators. In paper III we analyzed the function of a specific Mediator subcomplex. Mediator from mammalian cells has been isolated in two different forms, the larger TRAP/Mediator complex and the smaller PC2/CRSP complex. The TRAP/Mediator complex contains 4 additional proteins, TRAP230, TRAP240, Srb10 and Srb11, which are absent in PC2/CRSP. We developed a purification scheme for the larger form of the S. pombe Mediator using the so-called tandem affinity purification tag (TAP). Our new purification procedure allowed to identify a novel form of Mediator, which also contained homologues to TRAP230, TRAP240, Srb10 and Srb11, which we denoted the TRAP240/Mediator. In paper IV we reconstituted a pure in vitro system for RNA polymerase II dependent transcription. We purified S. pombe general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enabled highly purified RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with S. cerevisiae TBP. We used the in vitro system to compare the activities of Mediator and the larger TRAP240/Mediator on basal transcription. We found that the smaller form of Mediator was able to stimulate transcription whereas the larger TRAP240/Mediator repressed transcription. Our studies lead us to propose a model for how the two forms of Mediator interact to regulate RNA polymerase II dependent transcription