32 research outputs found
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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
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
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
RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing
Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing
Distinct pre-initiation steps in human mitochondrial translation
Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mamma
LRPPRC-mediated folding of the mitochondrial transcriptome
The mitochondrial genome, being compressed to 16âkb, is an attractive model system to investigate how RNA-binding proteins chaperone mRNA lifecycles. Here the authors use RNase footprinting and PAR-CLIP to show that the LRPPRCâSLIRP complex stabilizes mRNA structures to expose sites required for translation and polyadenylation
Structure of the human MTERF4âNSUN4 protein complex that regulates mitochondrial ribosome biogenesis
Disease causing mutation (P178L) in mitochondrial transcription factor A results in impaired mitochondrial transcription initiation
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Mitochondrial translation and ribosome assembly in <i>Nsun4</i> knockout hearts.
<p>A. Pulse-labeling of mitochondrial translation products in isolated heart mitochondria from 20 weeks-old control (L/L) and knockout mice (L/L, cre). The Coomassie-stained gel is a loading control. Known mitochondrial polypeptides are indicated. B. Western blot analysis of steady-state levels of mitochondrially and nucleus-encoded OXPHOS proteins in mitochondria from control and knockout hearts at different ages. VDAC was used as a loading control. *, cross reaction. C. Analysis of mitoribosomal assembly by sucrose gradient ultracentrifugation of heart mitochondrial extracts from control (L/L) and mutant (L/L, cre) mice. Sedimentation of 28S (SSU, fraction 6), 39S (LSU, fraction 8) and 55S (assembled ribosomes, fraction 10) was determined by western blot analysis using MRPS15- and MRPL13-specific antibodies. D. Western blot analysis of steady-state levels of MRPL13 and MRPS15 in heart mitochondrial extracts from control (L/L) and knockout (L/L, cre) mice at different ages. VDAC was used as a loading control.</p