Nova Reader - Subject https://www.novareader.co Default RSS Feed en-us Newgen KnowledgeWorks <![CDATA[Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab228 Deficient maturations of mitochondrial transcripts are linked to clinical abnormalities but their pathophysiology remains elusive. Previous investigations showed that pathogenic variants in MTO1 for the biosynthesis of τm5U of tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR) were associated with hypertrophic cardiomyopathy (HCM). Using mto1 knock-out(KO) zebrafish generated by CRISPR/Cas9 system, we demonstrated the pleiotropic effects of Mto1 deficiency on mitochondrial RNA maturations. The perturbed structure and stability of tRNAs caused by mto1 deletion were evidenced by conformation changes and sensitivity to S1-mediated digestion of tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). Notably, mto1KO zebrafish exhibited the global decreases in the aminoacylation of mitochondrial tRNAs with the taurine modification. Strikingly, ablated mto1 mediated the expression of MTPAP and caused the altered polyadenylation of cox1, cox3, and nd1 mRNAs. Immunoprecipitation assay indicated the interaction of MTO1 with MTPAP related to mRNA polyadenylation. These alterations impaired mitochondrial translation and reduced activities of oxidative phosphorylation complexes. These mitochondria dysfunctions caused heart development defects and hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the mto1KO zebrafish recapitulated the clinical phenotypes in HCM patients carrying the MTO1 mutation(s). Our findings highlighted the critical role of MTO1 in mitochondrial transcript maturation and their pathological consequences in hypertrophic cardiomyopathy.

]]> <![CDATA[Eukaryote specific RNA and protein features facilitate assembly and catalysis of H/ACA snoRNPs]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab177 H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.

]]> <![CDATA[ <i>Escherichia coli</i> RNase E can efficiently replace RNase Y in <i>Bacillus subtilis</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab216 RNase Y and RNase E are disparate endoribonucleases that govern global mRNA turnover/processing in the two evolutionary distant bacteria Bacillus subtilis and Escherichia coli, respectively. The two enzymes share a similar in vitro cleavage specificity and subcellular localization. To evaluate the potential equivalence in biological function between the two enzymes in vivo we analyzed whether and to what extent RNase E is able to replace RNase Y in B. subtilis. Full-length RNase E almost completely restores wild type growth of the rny mutant. This is matched by a surprising reversal of transcript profiles both of individual genes and on a genome-wide scale. The single most important parameter to efficient complementation is the requirement for RNase E to localize to the inner membrane while truncation of the C-terminal sequences corresponding to the degradosome scaffold has only a minor effect. We also compared the in vitro cleavage activity for the major decay initiating ribonucleases Y, E and J and show that no conclusions can be drawn with respect to their activity in vivo. Our data confirm the notion that RNase Y and RNase E have evolved through convergent evolution towards a low specificity endonuclease activity universally important in bacteria.

]]> <![CDATA[Coevolution of ribosomal RNA expansion segment 7<sup>L</sup> and assembly factor Noc2p specializes the ribosome biogenesis pathway between <i>Saccharomyces cerevisiae</i> and <i>Candida albicans</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab218 Ribosomes of different species share an evolutionarily conserved core, exhibiting flexible shells formed partially by the addition of species-specific ribosomal RNAs (rRNAs) with largely unexplored functions. In this study, we showed that by swapping the Saccharomyces cerevisiae 25S rRNA genes with non-S. cerevisiae homologs, species-specific rRNA variations caused moderate to severe pre-rRNA processing defects. Specifically, rRNA substitution by the Candida albicans caused severe growth defects and deficient pre-rRNA processing. We observed that such defects could be attributed primarily to variations in expansion segment 7L (ES7L) and could be restored by an assembly factor Noc2p mutant (Noc2p-K384R). We showed that swapping ES7L attenuated the incorporation of Noc2p and other proteins (Erb1p, Rrp1p, Rpl6p and Rpl7p) into pre-ribosomes, and this effect could be compensated for by Noc2p-K384R. Furthermore, replacement of Noc2p with ortholog from C. albicans could also enhance the incorporation of Noc2p and the above proteins into pre-ribosomes and consequently restore normal growth. Taken together, our findings help to elucidate the roles played by the species-specific rRNA variations in ribosomal biogenesis and further provide evidence that coevolution of rRNA expansion segments and cognate assembly factors specialized the ribosome biogenesis pathway, providing further insights into the function and evolution of ribosome.

]]> <![CDATA[Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab242 Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.

]]> <![CDATA[A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77<sup>Gag</sup>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab223 Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

A single stranded purines (ssPurine) loop in a bifurcated stem-loop structure containing the genomic RNA dimerization initiation site (DIS) hairpin, and the primer binding site (PBS) play a crucial role in the packaging of MMTV genomic RNA by recruiting Pr77Gag precursor polyprotein.
Graphical Abstract
A single stranded purines (ssPurine) loop in a bifurcated stem-loop structure containing the genomic RNA dimerization initiation site (DIS) hairpin, and the primer binding site (PBS) play a crucial role in the packaging of MMTV genomic RNA by recruiting Pr77Gag precursor polyprotein.

]]> <![CDATA[Profiling of RNA ribose methylation in <i>Arabidopsis thaliana</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab196 Eukaryotic rRNAs and snRNAs are decorated with abundant 2′-O-methylated nucleotides (Nm) that are predominantly synthesized by box C/D snoRNA-guided enzymes. In the model plant Arabidopsis thaliana, C/D snoRNAs have been well categorized, but there is a lack of systematic mapping of Nm. Here, we applied RiboMeth-seq to profile Nm in cytoplasmic, chloroplast and mitochondrial rRNAs and snRNAs. We identified 111 Nm in cytoplasmic rRNAs and 19 Nm in snRNAs and assigned guide for majority of the detected sites using an updated snoRNA list. At least four sites are directed by guides with multiple specificities as shown in yeast. We found that C/D snoRNAs frequently form extra pairs with nearby sequences of methylation sites, potentially facilitating the substrate binding. Chloroplast and mitochondrial rRNAs contain five almost identical methylation sites, including two novel sites mediating ribosomal subunit joining. Deletion of FIB1 or FIB2 gene reduced the accumulation of C/D snoRNA and rRNA methylation with FIB1 playing a bigger role in methylation. Our data reveal the comprehensive 2′-O-methylation maps for Arabidopsis rRNAs and snRNAs and would facilitate study of their function and biosynthesis.

]]> <![CDATA[RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2′-<i>O</i>-methylation]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab159 RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2′-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and ‘free’ pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2′-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.

]]> <![CDATA[Computational meta-analysis of ribosomal RNA fragments: potential targets and interaction mechanisms]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab190 The most abundant cellular RNA species, ribosomal RNA (rRNA), appears to be a source of massive amounts of non-randomly generated fragments. We found rRNA fragments (rRFs) in immunoprecipitated Argonaute (Ago-IP) complexes in human and mouse cells and in small RNA sequencing datasets. In human Ago1-IP, guanine-rich rRFs were preferentially cut in single-stranded regions of mature rRNAs between pyrimidines and adenosine, and non-randomly paired with cellular transcripts in crosslinked chimeras. Numerous identical rRFs were found in the cytoplasm and nucleus in mouse Ago2-IP. We report specific interaction motifs enriched in rRF-target pairs. Locations of such motifs on rRFs were compatible with the Ago structural features and patterns of the Ago-RNA crosslinking in both species. Strikingly, many of these motifs may bind to double-stranded regions on target RNAs, suggesting a potential pathway for regulating translation by unwinding mRNAs. Occurring on either end of rRFs and matching intronic, untranslated or coding regions in targets, such interaction sites extend the concept of microRNA seed regions. Targeting both borders of certain short introns, rRFs may be involved in their biogenesis or function, facilitated by Ago. Frequently dismissed as noise, rRFs are poised to greatly enrich the known functional spectrum of small RNA regulation.

]]> <![CDATA[A RanBP2-type zinc finger protein functions in intron splicing in Arabidopsis mitochondria and is involved in the biogenesis of respiratory complex I]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab066 The RanBP2 zinc finger (Znf) domain is a prevalent domain that mediates protein interaction and RNA binding. In Arabidopsis, a clade of four RanBP2 Znf-containing proteins, named the Organelle Zinc (OZ) finger family, are known or predicted to be targeted to either the mitochondria or the plastids. Previously we reported that OZ1 is absolutely required for the editing of 14 sites in chloroplasts. We now have investigated the function of OZ2, whose null mutation is embryo lethal. We rescued the null mutant by expressing wild-type OZ2 under the control of the seed-specific ABSCISIC ACID-INSENSITIVE3 (ABI3) promoter. Rescued mutant plants exhibit severely delayed development and a distinctive morphological phenotype. Genetic and biochemical analyses demonstrated that OZ2 promotes the splicing of transcripts of several mitochondrial nad genes and rps3. The splicing defect of nad transcripts results in the destabilization of complex I, which in turn affects the respiratory ability of oz2 mutants, turning on the alternative respiratory pathway, and impacting the plant development. Protein-protein interaction assays demonstrated binding of OZ2 to several known mitochondrial splicing factors targeting the same splicing events. These findings extend the known functional repertoire of the RanBP2 zinc finger domain in nuclear splicing to include plant organelle splicing.

]]> <![CDATA[Disruption of exon-bridging interactions between the minor and major spliceosomes results in alternative splicing around minor introns]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab118 Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry–Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.

]]> <![CDATA[Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab129 Uridine insertion/deletion editing of mitochondrial mRNAs is a characteristic feature of kinetoplastids, including Trypanosoma brucei. Editing is directed by trans-acting gRNAs and catalyzed by related RNA Editing Core Complexes (RECCs). The non-catalytic RNA Editing Substrate Binding Complex (RESC) coordinates interactions between RECC, gRNA and mRNA. RESC is a dynamic complex comprising GRBC (Guide RNA Binding Complex) and heterogeneous REMCs (RNA Editing Mediator Complexes). Here, we show that RESC10 is an essential, low abundance, RNA binding protein that exhibits RNase-sensitive and RNase-insensitive interactions with RESC proteins, albeit its minimal in vivo interaction with RESC13. RESC10 RNAi causes extensive RESC disorganization, including disruption of intra-GRBC protein–protein interactions, as well as mRNA depletion from GRBC and accumulation on REMCs. Analysis of mitochondrial RNAs at single nucleotide resolution reveals transcript-specific effects: RESC10 dramatically impacts editing progression in pan-edited RPS12 mRNA, but is critical for editing initiation in mRNAs with internally initiating gRNAs, pointing to distinct initiation mechanisms for these RNA classes. Correlations between sites at which editing pauses in RESC10 depleted cells and those in knockdowns of previously studied RESC proteins suggest that RESC10 acts upstream of these factors and that RESC is particularly important in promoting transitions between uridine insertion and deletion RECCs.

]]> <![CDATA[A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab123 CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

]]> <![CDATA[Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab116 Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3′ untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.

]]> <![CDATA[mTORC1 promotes TOP mRNA translation through site-specific phosphorylation of LARP1]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1239 LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5′TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1’s translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 ‘pendular hook’ engages the TOP mRNA 5′-end to repress translation, but only in conditions of mTORC1 inhibition.

]]> <![CDATA[Another layer of complexity in <i>Staphylococcus aureus</i> methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines <i>met</i> operon mRNA stability and decay]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1277 In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.

The MET-T-box riboswitch, controlling methionine biosynthesis in Staphylococcus aureus, is target of RNase III-mediated cleavage which in turn initiates met operon mRNA decay from the 5′-end involving RNase J.
Graphical Abstract
The MET-T-box riboswitch, controlling methionine biosynthesis in Staphylococcus aureus, is target of RNase III-mediated cleavage which in turn initiates met operon mRNA decay from the 5′-end involving RNase J.

]]> <![CDATA[Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab017 Ribosome hibernation is a universal translation stress response found in bacteria as well as plant plastids. The term was coined almost two decades ago and despite recent insights including detailed cryo-EM structures, the physiological role and underlying molecular mechanism of ribosome hibernation has remained unclear. Here, we demonstrate that Escherichia coli hibernation factors RMF, HPF and RaiA (HFs) concurrently confer ribosome hibernation. In response to carbon starvation and resulting growth arrest, we observe that HFs protect ribosomes at the initial stage of starvation. Consistently, a deletion mutant lacking all three factors (ΔHF) is severely inhibited in regrowth from starvation. ΔHF cells increasingly accumulate 70S ribosomes harbouring fragmented rRNA, while rRNA in wild-type 100S dimers is intact. RNA fragmentation is observed to specifically occur at HF-associated sites in 16S rRNA of assembled 70S ribosomes. Surprisingly, degradation of the 16S rRNA 3′-end is decreased in cells lacking conserved endoribonuclease YbeY and exoribonuclease RNase R suggesting that HFs directly block these ribonucleases from accessing target sites in the ribosome.

]]> <![CDATA[Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA–protein complexes]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab008 A set of >300 nonredundant high-resolution RNA–protein complexes were rigorously searched for π-contacts between an amino acid side chain (W, H, F, Y, R, E and D) and an RNA nucleobase (denoted π–π interaction) or ribose moiety (denoted sugar–π). The resulting dataset of >1500 RNA–protein π-contacts were visually inspected and classified based on the interaction type, and amino acids and RNA components involved. More than 80% of structures searched contained at least one RNA–protein π-interaction, with π–π contacts making up 59% of the identified interactions. RNA–protein π–π and sugar–π contacts exhibit a range in the RNA and protein components involved, relative monomer orientations and quantum mechanically predicted binding energies. Interestingly, π–π and sugar–π interactions occur more frequently with RNA (4.8 contacts/structure) than DNA (2.6). Moreover, the maximum stability is greater for RNA–protein contacts than DNA–protein interactions. In addition to highlighting distinct differences between RNA and DNA–protein binding, this work has generated the largest dataset of RNA–protein π-interactions to date, thereby underscoring that RNA–protein π-contacts are ubiquitous in nature, and key to the stability and function of RNA–protein complexes.

]]> <![CDATA[Targeted splice sequencing reveals RNA toxicity and therapeutic response in myotonic dystrophy]]> https://www.novareader.co/book/isbn/10.1093/nar/gkab022 Biomarker-driven trials hold promise for therapeutic development in chronic diseases, such as muscular dystrophy. Myotonic dystrophy type 1 (DM1) involves RNA toxicity, where transcripts containing expanded CUG-repeats (CUGexp) accumulate in nuclear foci and sequester splicing factors in the Muscleblind-like (Mbnl) family. Oligonucleotide therapies to mitigate RNA toxicity have emerged but reliable measures of target engagement are needed. Here we examined muscle transcriptomes in mouse models of DM1 and found that CUGexp expression or Mbnl gene deletion cause similar dysregulation of alternative splicing. We selected 35 dysregulated exons for further study by targeted RNA sequencing. Across a spectrum of mouse models, the individual splice events and a composite index derived from all events showed a graded response to decrements of Mbnl or increments of CUGexp. Antisense oligonucleotides caused prompt reduction of CUGexp RNA and parallel correction of the splicing index, followed by subsequent elimination of myotonia. These results suggest that targeted splice sequencing may provide a sensitive and reliable way to assess therapeutic impact in DM1.

]]> <![CDATA[Ecd promotes U5 snRNP maturation and Prp8 stability]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1274 Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes.

]]> <![CDATA[Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1268 Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

]]> <![CDATA[tRNA 3′ shortening by LCCR4 as a response to stress in <i>Trypanosoma brucei</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1261 Sensing of environmental cues is crucial for cell survival. To adapt to changes in their surroundings cells need to tightly control the repertoire of genes expressed at any time. Regulation of translation is key, especially in organisms in which transcription is hardly controlled, like Trypanosoma brucei. In this study, we describe the shortening of the bulk of the cellular tRNAs during stress at the expense of the conserved 3′ CCA-tail. This tRNA shortening is specific for nutritional stress and renders tRNAs unsuitable substrates for translation. We uncovered the nuclease LCCR4 (Tb927.4.2430), a homologue of the conserved deadenylase Ccr4, as being responsible for tRNA trimming. Once optimal growth conditions are restored tRNAs are rapidly repaired by the trypanosome tRNA nucleotidyltransferase thus rendering the recycled tRNAs amenable for translation. This mechanism represents a fast and efficient way to repress translation during stress, allowing quick reactivation with a low energy input.

]]> <![CDATA[NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1226 The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.

]]> <![CDATA[Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1260 Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.

]]> <![CDATA[A deafness-associated tRNA mutation caused pleiotropic effects on the m<sup>1</sup>G37 modification, processing, stability and aminoacylation of tRNA<sup>Ile</sup> and mitochondrial translation]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1225 Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3′ adjacent to the tRNA’s anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5′ end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.

]]> <![CDATA[Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions <i>in vivo</i>]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1244 The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

]]> <![CDATA[The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1189 The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5′ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.

]]> <![CDATA[Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1178 Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 ‘writes’ modifications on tRNA and mRNA, and both types of RNAs are ‘read’ by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.

Pseudouridylation by Pus6 enhances MetRS binding of tRNA and mRNA, to confer global and gene-specific translation regulation, respectively.
Graphical Abstract
Pseudouridylation by Pus6 enhances MetRS binding of tRNA and mRNA, to confer global and gene-specific translation regulation, respectively.

]]> <![CDATA[Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1187 In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.

]]> <![CDATA[Multiple competing RNA structures dynamically control alternative splicing in the human ATE1 gene]]> https://www.novareader.co/book/isbn/10.1093/nar/gkaa1208 The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1–R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.

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