Prokaryotic RNA N1-Methyladenosine Erasers Maintain tRNA m1A Modification Levels in Streptomyces venezuelae.

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, m1A tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. M1A in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its m1A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified that acts on endogenous RNA modifications. Using Streptomyces venezuelae as a model organism, we confirmed the presence and quantitative m1A tRNA signatures using mass spectrometry and high-throughput tRNA sequencing. We identified two RNA demethylases that can remove m1A in tRNA and validated the activity of a previously annotated tRNA m1A writer. Using single-gene knockouts of these erasers and the m1A writer, we found dynamic changes of m1A levels in many tRNAs under stress conditions. Phenotypic characterization highlighted changes in their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms of translational regulation in bacteria.

Principles of miRNA/miRNA* function in plant MIRNA processing.

MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

The DEAD-box RNA helicase RhlB is required for efficient RNA processing at low temperature in Caulobacter.

IMPORTANCE: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.

The nucleolus of Giardia and its ribosomal biogenesis.

Giardia duodenalis is a protozoan intestinal parasite that causes a significant number of infections worldwide each year, particularly in low-income and developing countries. Despite the availability of treatments for this parasitic infection, treatment failures are alarmingly common. As a result, new therapeutic strategies are urgently needed to effectively combat this disease. On the other hand, within the eukaryotic nucleus, the nucleolus stands out as the most prominent structure. It plays a crucial role in coordinating ribosome biogenesis and is involved in vital processes such as maintaining genome stability, regulating cell cycle progression, controlling cell senescence, and responding to stress. Given its significance, the nucleolus presents itself as a valuable target for selectively inducing cell death in undesirable cells, making it a potential avenue for anti-Giardia treatments. Despite its potential importance, the Giardia nucleolus remains poorly studied and often overlooked. In light of this, the objective of this study is to provide a detailed molecular description of the structure and function of the Giardia nucleolus, with a primary focus on its involvement in ribosomal biogenesis. Likewise, it discusses the targeting of the Giardia nucleolus as a therapeutic strategy, its feasibility, and the challenges involved.

Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system.

Trypanosoma brucei belongs to a group of protozoans presenting fragmented large subunit rRNA. Its LSU rRNA equivalent to the 25S/28S rRNA of other eukaryotes is split into six fragments, requiring additional processing for removal of the extra spacer sequences. We have used a genetic complementation strategy to further investigate the T. brucei RRP44 nuclease in pre-rRNA maturation. TbRRP44 contains both a PIN and a RNB domain whose homologues are found in association with the exosome complex. We found that the exonucleolytic activity of the RNB domain as well as the physical presence of the PIN domain are essential for TbRRP44 function, while a catalytic site mutation in the PIN domain has no detectable effect on cell growth. A new endonucleolytic cleavage site in ITS1 was identified. In addition to the 5.8S rRNA 3'-end maturation, TbRRP44 is required for degradation of the excised 5'-ETS and for removal of part of ITS1 during maturation of the 18S rRNA 3'-end. TbRRP44 deficiency leads to accumulation of many LSU intermediate precursors, most of them not detected in control cells. TbRRP44 is also required for U3 snoRNA and spliced leader processing, indicating that TbRRP44 may have a wide role in RNA processing in T. brucei.

Toward a systems view on RNA-binding proteins and associated RNAs in plants: Guilt by association.

RNA-binding proteins (RBPs) have a broad impact on most biochemical, physiological, and developmental processes in a plant's life. RBPs engage in an on-off relationship with their RNA partners, accompanying virtually every stage in RNA processing and function. While the function of a plethora of RBPs in plant development and stress responses has been described, we are lacking a systems-level understanding of components in RNA-based regulation. Novel techniques have substantially enlarged the compendium of proteins with experimental evidence for binding to RNAs in the cell, the RNA-binding proteome. Furthermore, ribonomics methods have been adapted for use in plants to profile the in vivo binding repertoire of RBPs genome-wide. Here, we discuss how recent technological achievements have provided novel insights into the mode of action of plant RBPs at a genome-wide scale. Furthermore, we touch upon two emerging topics, the connection of RBPs to phase separation in the cell and to extracellular RNAs. Finally, we define open questions to be addressed to move toward an integrated understanding of RBP function.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants.

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

Evidence for reduced plasmodesmata callose accumulation in Nicotiana benthamiana leaves with increased symplastic cell-to-cell communication caused by RNA processing defects of chloroplasts.

RNA processing defects in chloroplasts were previously associated with increased plasmodesmata (PD) permeability. However, the underlying mechanisms for such association are still unknown. To provide insight into this, we silenced the expression of chloroplast-located INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) RNA helicase in Nicotiana benthamiana leaves and determined an increase in PD permeability which is caused by a reduction of PD callose deposition. Moreover, the silencing of two other nuclear genes encoding chloroplastic enzymes involved in RNA processing, RH3, and CLPR2, also increased PD permeability accompanied by reduced callose accumulation at PD. In addition, we quantified the plastidic hydrogen peroxide levels using the chloroplast-targeted fluorescent sensor, HyPer, in ISE2, RH3, and CLPR2 silenced N. benthamiana leaves. The levels of chloroplastic hydrogen peroxide were not correlated with the increased cell-to-cell movement of the marker protein GFP2X. We, therefore, propose that defects in chloroplast RNA metabolism mediate PD gating by suppressing PD callose deposition, and hydrogen peroxide levels in the organelles are not directly linked to this process.

Post-transcriptional diversity in riboproteins and RNAs in aging and cancer.

Post-transcriptional (PtscM) and post-translational (PtrnM) modifications of nucleotides and amino acids are covalent modifications able to change physio-chemical properties of RNAs and proteins. In the ribosome, the adequate assembly of rRNAs and ribosomal protein subunits in the nucleolus ensures suitable translational activity, with protein synthesis tuned according to intracellular demands of energy production, replication, proliferation, and growth. Disruption in the regulatory control of PtscM and PtrnM can impair ribosome biogenesis and ribosome function. Ribosomal impairment may, in turn, impact the synthesis of proteins engaged in functions as varied as telomere maintenance, apoptosis, and DNA repair, as well as intersect with mitochondria and telomerase activity. These cellular processes often malfunction in carcinogenesis and senescence. Here we discuss regulatory mechanisms of PtscMs and PtrnMs on ribosomal function. We also address chemical modification in rRNAs and their impacts on cellular metabolism, replication control, and senescence. Further, we highlight similarities and differences of PtscMs and PtrnMs in ribosomal intermediates during aging and carcinogenesis. Understanding these regulatory mechanisms may uncover critical steps for the development of more efficient oncologic and anti-aging therapies.

Physio-pathological effects of m6A modification and its potential contribution to melanoma.

Methylation of N6-adenosine (m6A) is the most prevalent internal RNA modification and is especially common among the messenger RNAs. These m6A modifications regulate splicing, translocation, stability and translation of RNA through dynamic and reversible interactions with m6A-binding proteins, namely the writers, erasers and readers. RNA methyltransferases catalyze the m6A modifications, while demethylases reverse this methylation. Deregulation of the m6A modification process has been implicated in human carcinogenesis, including melanoma-which carries one of the highest mutant rates. In this review, we provide an up-to-date summary of m6A regulation and its biological impacts on normal and cancer cells, with emphasis on the deregulation of m6A modification and m6A regulators in melanoma. In addition, we highlight the prospective potential of exploiting m6A modification in the treatment of melanoma and non-cancer diseases.

The ribosome assembly factor Nop53 has a structural role in the formation of nuclear pre-60S intermediates, affecting late maturation events.

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S 'foot'. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome.

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5' splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Epitranscriptome machinery in Trypanosomatids: New players on the table?

Trypanosoma and Leishmania parasites cause devastating tropical diseases resulting in serious global health consequences. These organisms have complex life cycles with mammalian hosts and insect vectors. The parasites must, therefore, survive in different environments, demanding rapid physiological and metabolic changes. These responses depend upon regulation of gene expression, which primarily occurs posttranscriptionally. Altering the composition or conformation of RNA through nucleotide modifications is one posttranscriptional mechanism of regulating RNA fate and function, and modifications including N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytidine (m5C), N4-acetylcytidine (ac4C), and pseudouridine (Ψ), dynamically regulate RNA stability and translation in diverse organisms. Little is known about RNA modifications and their machinery in Trypanosomatids, but we hypothesize that they regulate parasite gene expression and are vital for survival. Here, we identified Trypanosomatid homologs for writers of m1A, m5C, ac4C, and Ψ and analyze their evolutionary relationships. We systematically review the evidence for their functions and assess their potential use as therapeutic targets. This work provides new insights into the roles of these proteins in Trypanosomatid parasite biology and treatment of the diseases they cause and illustrates that Trypanosomatids provide an excellent model system to study RNA modifications, their molecular, cellular, and biological consequences, and their regulation and interplay.

HASTY modulates miRNA biogenesis by linking pri-miRNA transcription and processing.

Post-transcriptional gene silencing mediated by microRNAs (miRNAs) modulates numerous developmental and stress response pathways. For the last two decades, HASTY (HST), the ortholog of human EXPORTIN 5, was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm. Here, we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis. We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs. Interestingly, protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain. Moreover, mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37. Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex, which in turn promotes the transcription and proper processing of primary miRNA transcripts (pri-miRNAs). Taken together, these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes, promoting the transcription and processing of pri-miRNAs rather than the direct export of processed miRNAs from the nucleus.

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach.

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2'-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2'-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

A birds'-eye view of the activity and specificity of the mRNA m6 A methyltransferase complex.

Appropriate control of the transcriptome is essential to regulate different aspects of gene expression during development and in response to environmental stimuli. Fast accumulating reports are recognizing and functionally characterizing several types of modifications across transcripts, which have created a new field of RNA study named epitranscriptomics. The most abundant modification found in messenger RNA (mRNA) is N6-methyladenosine (m6 A). m6 A addition is achieved by a large methyltransferase complex (MTC). The m6 A-MTC is composed of the methyltransferases METTL3 and METTL14 as the catalytic core, and several protein factors necessary for its correct catalysis, which include WTAP, RBM15, VIRMA, HAKAI, and ZC3H13. To fully appreciate the relevance of this modification, it is important to dissect the basis for the MTC function as well as to define its interaction with other cellular partners. Here, we summarize previous and recent knowledge on these issues to provide a guide for future research and put forward ideas on the flexibility and specificity of this process. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition.

Non-coding RNAs repressive role in post-transcriptional processing of RUNX2 during the acquisition of the osteogenic phenotype of periodontal ligament mesenchymal stem cells.

Mesenchymal stem cells are candidates for therapeutic strategies in periodontal repair due to their osteogenic potential. In this study, we identified epigenetic markers during osteogenic differentiation, taking advantage of the individual pattern of mesenchymal cells of the periodontal ligament with high (h-PDLCs) and low (l-PDLCs) osteogenic capacity. We found that the involvement of non-coding RNAs in the regulation of the RUNX2 gene is strongly associated with high osteogenic potential. Moreover, we evaluated miRs and genes that encode enzymes to process miRs and their biogenesis. Our data show the high expression of the XPO5 gene, and miRs 7 and 22 observed in the l-PDLCs might be involved in acquiring osteogenic potential, suppressing RUNX2 gene expression. Further, an inversely proportional correlation between lncRNAs (HOTAIR and HOTTIP) and RUNX2 gene expression was observed in both l- and h-PDLCs, and it was also related to the distinct osteogenic phenotypes. Thus, our results indicate the low expression of XPO5 in h-PDLC might be the limiting point for blocking the miRs biogenesis, allowing the high gene expression of RUNX2. In accordance, the low expression of miRs, HOTAIR, and HOTTIP could be a prerequisite for increased osteogenic potential in h-PDLCs. These results will help us to better understand the underlying mechanisms of osteogenesis, considering the heterogeneity in the osteogenic potential of PDLCs that might be related to a distinct transcriptional profile of lncRNAs and the biogenesis machinery.

Advances in the Bioinformatics Knowledge of mRNA Polyadenylation in Baculovirus Genes.

Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3' end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3' ends from 180 baculovirus genomes.

Identification of key sequence features required for microRNA biogenesis in plants.

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.