RNase Z is an endonuclease responsible for the removal of 3' extensions from tRNA precursors, an essential step in tRNA biogenesis. Human cells contain a long form (RNase Z(L)) encoded by ELAC2, and ...a short form (RNase Z(S); ELAC1). We studied their subcellular localization by expression of proteins fused to green fluorescent protein. RNase Z(S) was found in the cytosol, whereas RNase Z(L) localized to the nucleus and mitochondria. We show that alternative translation initiation is responsible for the dual targeting of RNase Z(L). Due to the unfavorable context of the first AUG of ELAC2, translation apparently also starts from the second AUG, whereby the mitochondrial targeting sequence is lost and the protein is instead routed to the nucleus. Our data suggest that RNase Z(L) is the enzyme involved in both, nuclear and mitochondrial tRNA 3' end maturation.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify ...m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%–25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is “hard coded” in cis via a simple and predictable code, accounting for 33%–46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.
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•RNA digestion via m6A sensitive RNase (MAZTER-seq) allows systematic m6A quantitation•MAZTER-seq reveals that antibody-based methods are of limited sensitivity•m6A stoichiometry is “hard coded” by a simple, predictable, and conserved code•MAZTER-seq allows quantitative tracking of m6A in diverse biological settings
A new enzymatic approach for precise mapping and measurement of m6A within mRNAs provides insight into how methylation sites are selected and the functional impact of the modifications.
Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N
-methyladenosine (m
A), which disrupts Watson-Crick ...base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m
A at single-nucleotide resolution. Within the cytosol, m
A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m
A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m
A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m
A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m
A levels was adopted as a potential means of post-transcriptional regulation.
Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial ...in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5' extensions, is the methyltransferase responsible for m(1)G9 and m(1)A9 formation. The ability of the mitochondrial tRNA:m(1)R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5' end processing.
Abstract
The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of ...which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.
tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5′ ends are removed by an endonuclease called RNase P. All RNase P enzymes ...characterized so far are composed of an RNA plus one or more proteins, and tRNA 5′ end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a
trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.
N6-methyladenosine (m6A) is the most abundant mRNA modification, and controls mRNA stability. m6A distribution varies considerably between and within species. Yet, it is unclear to what extent this ...variability is driven by changes in genetic sequences ('cis') or cellular environments ('trans') and via which mechanisms.
Here we dissect the determinants governing RNA methylation via interspecies and intraspecies hybrids in yeast and mammalian systems, coupled with massively parallel reporter assays and m6A-QTL reanalysis. We find that m6A evolution and variability is driven primarily in 'cis', via two mechanisms: (1) variations altering m6A consensus motifs, and (2) variation impacting mRNA secondary structure. We establish that mutations impacting RNA structure - even when distant from an m6A consensus motif - causally dictate methylation propensity. Finally, we demonstrate that allele-specific differences in m6A levels lead to allele-specific changes in gene expression.
Our findings define the determinants governing m6A evolution and diversity and characterize the consequences thereof on gene expression regulation.
Of P and Z: Mitochondrial tRNA processing enzymes Rossmanith, Walter
Biochimica et biophysica acta,
September-October 2012, 2012 Sep-Oct, 2012-09-00, 20120901, Letnik:
1819, Številka:
9-10
Journal Article
Recenzirano
Odprti dostop
Mitochondrial tRNAs are generally synthesized as part of polycistronic transcripts. Release of tRNAs from these precursors is thus not only required to produce functional adaptors for translation, ...but also responsible for the maturation of other mitochondrial RNA species. Cleavage of mitochondrial tRNAs appears to be exclusively accomplished by endonucleases. 5′-end maturation in the mitochondria of different Eukarya is achieved by various kinds of RNase P, representing the full range of diversity found in this enzyme family. While ribonucleoprotein enzymes with RNA components of bacterial-like appearance are found in a few unrelated protists, algae, and fungi, highly degenerate RNAs of dramatic size variability are found in the mitochondria of many fungi. The majority of mitochondrial RNase P enzymes, however, appear to be pure protein enzymes. Human mitochondrial RNase P, the first to be identified and possibly the prototype of all animal mitochondrial RNases P, is composed of three proteins. Homologs of its nuclease subunit MRPP3/PRORP, are also found in plants, algae and several protists, where they are apparently responsible for RNase P activity in mitochondria (and beyond) without the help of extra subunits. The diversity of RNase P enzymes is contrasted by the uniformity of mitochondrial RNases Z, which are responsible for 3′-end processing. Only the long form of RNase Z, which is restricted to eukarya, is found in mitochondria, even when an additional short form is present in the same organism. Mitochondrial tRNA processing thus appears dominated by new, eukaryal inventions rather than bacterial heritage. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.
► A great diversity of RNase P enzymes is found in mitochondria of different organisms. ► The RNase P of many fungi contains a highly degenerated RNA. ► Most mitochondrial RNase P enzymes, however, are composed of protein only. ► Mitochondrial 3′-end processing seems to rely exclusively on the long, eukaryal form of RNase Z.
RNase P is the enzyme that removes 5' extensions from tRNA precursors. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ...ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5' or 3' extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship.
The amyloid-β peptide (Aβ) is suggested to cause mitochondrial dysfunction in Alzheimer's disease. The mitochondrial dehydrogenase SDR5C1 (also known as ABAD) was shown to bind Aβ and was proposed to ...thereby mediate mitochondrial toxicity, but the molecular mechanism has not been clarified. We recently identified SDR5C1 as an essential component of human mitochondrial RNase P and its associated tRNA:m¹R9 methyltransferase, the enzymes responsible for tRNA 5'-end processing and methylation of purines at tRNA position 9, respectively. With this work we investigated whether SDR5C1's role as a subunit of these two tRNA-maturation activities represents the mechanistic link between Aβ and mitochondrial dysfunction. Using recombinant enzyme components, we tested RNase P and methyltransferase activity upon titration of Aβ. Micromolar concentrations of monomeric or oligomerized Aβ were required to inhibit tRNA 5'-end processing and position 9 methylation catalyzed by the SDR5C1-containing enzymes, yet similar concentrations of Aβ also inhibited related RNase P and methyltransferase activities, which do not contain an SDR5C1 homolog. In conclusion, the proposed deleterious effect of Aβ on mitochondrial function cannot be explained by a specific inhibition of mitochondrial RNase P or its tRNA:m¹R9 methyltransferase subcomplex, and the molecular mechanism of SDR5C1-mediated Aβ toxicity remains unclear.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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