Substrate recognition and cleavage-site selection by a single-subunit protein-only RNase P

Brillante, Nadia; Gößringer, Markus; Lindenhofer, Dominik; Toth, Ursula; Rossmanith, Walter; Hartmann, Roland K.

doi:10.1093/nar/gkw080

Cited by 36 publications

(84 citation statements)

References 69 publications

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“…Though unlikely, we cannot rule out the possibility these conditions do not entirely reflect binding of substrates to mtRNase P in MgCl2, when cleavage can occur. The kobs values for these substrates range between 0.4 -0.8 min -1 ( Figure 3C, Table 1) and correlate well with rate constants previously measured for single-subunit PRORPs cleaving their native substrates Brillante et al 2016;Howard et al 2016). In contrast, (mt)pre-tRNAs that have low affinities for mtRNase P ((mt)pre-tRNA Val , (mt)pre-tRNASer UCN , (mt)pre-tRNA Ser(AGY) )) exhibit ~ 100-fold lower single turnover activity (kobs ~0.06 min -1 ) ( Figure 3C and Table 1); the cleavage of these substrates was so slow that we were unable to accurately measure K1/2 MRPP3 values.…”

Section: Mtrnase P Exhibits 5' End Processing Of Tightly Bound (Mt)prsupporting

confidence: 85%

“…The exact lengths of 5' and 3' flanking ends of mtRNase P substrates have not yet been established; therefore, we used our knowledge of A. thaliana single subunit PRORP enzymes that are homologous to MRPP3 to design the 5' and 3' ends of our (mt)pre-tRNA substrates. PRORPs preferentially bind substrates with short (< 5-8 nucleotide) 5' leaders and do not bind the 3' trailer of pre-tRNAs Howard et al 2016;Brillante et al 2016). Thus, the (mt)pre-tRNA substrates studied here possess 6-7 nucleotide long 5' leader sequences and lack mature 3' CCA ends (Figure 1).…”

Section: Mtrnase P Binds Mitochondrial Precursor-trnas Non-uniformlymentioning

confidence: 88%

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Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

Karasik

Fierke

Koutmos

2018

Preprint

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Human mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5' end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo.Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs.Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

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Section: Mtrnase P Exhibits 5' End Processing Of Tightly Bound (Mt)prsupporting

confidence: 85%

Section: Mtrnase P Binds Mitochondrial Precursor-trnas Non-uniformlymentioning

confidence: 88%

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

Karasik

Fierke

Koutmos

2018

Preprint

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“…As previously discussed, the biological significance of this feature is elusive and such acceptor stem extensions appear to be under negative selection in the nuclear tRNA genes of A. thaliana (34). Counterintuitively, A. thaliana mitochondria and chloroplasts encode bacterial-type tRNA His genes (http://plantrna.ibmp.cnrs.fr/; (41)), however the organellar At PRORP1 cleaved only <50% of E. coli pre-tRNA His at the upstream ‘bacterial-like’ site (–2/–1).…”

Section: Discussionmentioning

confidence: 99%

“…Such G:C-rich extensions are not found in precursor tRNAs of A. thaliana (34), indicating that At PRORP enzymes have not been under evolutionary pressure to cope with such substrates. This is also in line with the in vitro processing results, demonstrating less efficient At PRORP processing of pre-tRNA Sec relative to pre-tRNA His and pre-tRNA Gly in particular (Supplementary Table S7).…”

Section: Discussionmentioning

confidence: 99%

Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP isoenzymes

Gößringer

Lechner

Brillante

et al. 2017

Nucleic Acids Research

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The RNase P family comprises structurally diverse endoribonucleases ranging from complex ribonucleoproteins to single polypeptides. We show that the organellar (AtPRORP1) and the two nuclear (AtPRORP2,3) single-polypeptide RNase P isoenzymes from Arabidopsis thaliana confer viability to Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. RNA-Seq revealed that AtPRORP1, compared with bacterial RNase P or AtPRORP3, cleaves several precursor tRNAs (pre-tRNAs) aberrantly in E. coli. Aberrant cleavage by AtPRORP1 was mainly observed for pre-tRNAs that can form short acceptor-stem extensions involving G:C base pairs, including tRNAAsp(GUC), tRNASer(CGA) and tRNAHis. However, both AtPRORP1 and 3 were defective in processing of E. coli pre-tRNASec carrying an acceptor stem expanded by three G:C base pairs. Instead, pre-tRNASec was degraded, suggesting that tRNASec is dispensable for E. coli under laboratory conditions. AtPRORP1, 2 and 3 are also essentially unable to process the primary transcript of 4.5S RNA, a hairpin-like non-tRNA substrate processed by E. coli RNase P, indicating that PRORP enzymes have a narrower, more tRNA-centric substrate spectrum than bacterial RNA-based RNase P enzymes. The cells’ viability also suggests that the essential function of the signal recognition particle can be maintained with a 5΄-extended 4.5S RNA.

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“…Proteins with LAGLIDADG motif are involved in catalytic processes due to its similarity with group-1 intron maturases [18] and those with SMR domain are related to MutS2 family which participate in transcription or repair of chloroplast DNA [19]. PRORP (proteinaceous RNaseP) sub-class possess metallonuclease domain which are involved in processing of mitochondrial tRNA, for example arabidopsis PRORP3 protein [20]. The classical P motif when interspersed by L motifs (36 amino acids) and S motifs (31 amino acids) in triplets constitute PLS sub-class, wherein this ordered association could have variable number of S motif repeats [21].…”

Section: Introductionmentioning

confidence: 99%

Insights into PPR Gene Family in Cajanus Cajan and Other Legume Species

Kaur¹,

Verma²

2016

J Data Mining Genomics Proteomics

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PPR proteins comprises of several hundred members among land plants and govern a fascinating array of functions in organeller genomes that ranges from participation in stabilization of organeller transcripts, RNA editing to fertility restoration of CMS lines. Despite the availability of genome sequences of several legume species, comprehensive cataloguing of members of PPR gene family has not been carried out. In the current study, we identified 523, 830, 534, 816, 441 and 677 PPR proteins in Cajanus, Glycine, Phaseolus, Medicago, Vigna and Cicer genomes, respectively and their complete in silico categorization was undertaken to classify them into various sub-classes and their localization prediction. Chromosomal coordinates of 271 Cajanus PPR genes were predicted and their homologues were identified in 5 other legumes revealing extensive genome conservation. PPR genes of all 6 legume species were further probed to identify restorer of fertility-like PPRs (RFLs) on the basis of protein clustering and followed by homology searches to already known Rf-PPR genes. Seventy RFL PPR genes (P sub-class) were identified and were scrutinized by phylogenetic analysis which revealed extended similarity and common features shared by these RFLs across the species. Some of these RFL PPRs were present as small clusters in Glycine, Phaseolus, Vigna and Cicer genomes. This study has generated a knowledge base about PPR gene family in legumes and opens several avenues for future investigations into their molecular functions, evolutionary relationships and their potential in identifying markers to enable cloning of Rf genes.Citation: Kaur P, Verma M, Chaduvula PK, Saxena S, Baliyan N, et al. (2016) Due to certain limitations of demarking PPR motifs by classical system of categorization, PPR motifs has been redefined by Cheng et al. [15] whereby 10 PPR motif variants have been described and used to annotate PPR sequences in 109 genomes. Newly identified motif variants in PLS sub-class includes P1 and P2 motifs that differ from classical P motif in first helix; S2 (32 amino acid), SS (31 amino acid), E1 (34 amino acid) and E2 (34 amino acid) motif. This revision of PPR classification has provided a clearer picture of PPR structure and thus will provide new insights into their role in molecular and structural evolution.Most of these nuclear encoded PPRs carry N-terminal mitochondrial or chloroplast targeting sequence and are important contributors of various organellar post-transcriptional processes by virtue of their sequence specific RNA binding activity. Considering the array of functions governed by PPR proteins, identification and characterization of homologous and species specific PPR proteins in other plant species is critical for understanding the dynamics of nuclear cytoplasmic interactions.Cytoplasmic male sterility system is widely exploited/ phenomenon for hybrid seed production and has been extensively studied at molecular and biochemical level in various crops. Fertility restorers are an important component o...

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Substrate recognition and cleavage-site selection by a single-subunit protein-only RNase P

Cited by 36 publications

References 69 publications

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP isoenzymes

Insights into PPR Gene Family in Cajanus Cajan and Other Legume Species

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