ICTS

Despite the presence and biological role of D-amino acids, the fundamental issue of how proteins are made only with the L-chiral entities was largely ignored. Over the last two decades, it has become clearer as to how multiple ‘Chiral Checkpoints’ work in concert to avoid D-amino acids from getting incorporated into proteins (1, 2). Our recent work has shed light on how chiral proofreading systems have played critical roles in important evolutionary transitions (3, 4). In the first part of my talk, I will introduce the work that came out of the laboratories of two of the pioneers of nucleic acid research in the area of protein biosynthesis, Paul Berg and Donald Crothers, more than half a century back. Their work on the identification of D-aminoacyl-tRNA deacylase (DTD) and ‘Discriminator hypothesis’, respectively, were hugely ahead of their time and were partly against the general paradigm at that time. In both of the above works, the smallest and the only achiral amino acid turned out to be an outlier as DTD can act weakly on glycine charged tRNAs with a unique discriminator base of ‘Uracil’. This peculiar nature of glycine remained an enigma for nearly half a century. With a load of available information on the subject by the turn of the century, our work on ‘chiral proofreading’ mechanisms during protein biosynthesis serendipitously led us to revisit these findings. Our analysis has uncovered an unexpected connection between them that has implications for evolution of different eukaryotic life forms (5) and will be the focus of the second part of my talk.
Selected references:
1. Kuncha, S. K. et al., J. Biol. Chem. 2019 (Review article).
2. Kumar, P. et al. FEBS Letters 2022 (Review article).
3. Gogoi, J. et al. Sci. Adv. 2022.
4. Kumar, P. et al. Proc. Natl. Acad. Sci. (USA) 2023.
5. Kumar, P. and Sankaranarayanan, R. NAR 2024 (Critical Reviews and Perspective article).

A hallmark pathological feature of ALS and FTD is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. We have been systematically identifying cryptic splicing targets regulated by TDP-43 in human brain. We discovered that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we found that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function. We are currently exploring the function of UNC13A in ALS/FTD and characterizing several other novel cryptic splicing targets. Some of these represent powerful biomarkers and other ones might be therapeutic targets. We are also using genome wide approaches to identify genes that work with TDP-43 to regulate cryptic splicing. Many cryptic splicing events caused by TDP-43 loss lead to cryptic transcripts degraded by the RNA surveillance mechanism nonsense-mediated decay (NMD). Standard RNA-sequencing approaches miss many of these and we recently found a way to unmask them (by inhibiting NMD). In addition to cryptic splicing, we have also discovered loss of TDP-43 in FTD/ALS leads to widespread alternative polyadenylation changes, impacting expression of disease-relevant genes and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.

tRNAs serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. Besides this canonical role, tRNAs are involved in other processes like programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. With help of S. cerevisiae and T. brucei, we demonstrated that those that do, establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determines the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

Protein synthesis is a fundamentally important process that lies at the center of metabolic and stress response pathways in the cell. Protein synthesis comes at a high metabolic cost for the cell, and its mis-regulation is frequently implicated in disease. For this reason, the main steps of protein synthesis are subjected to rigorous quality control pathways that ensure efficiency, fidelity, and homeostasis. A widespread strategy employed by pathogens to establish infection is to inhibit host protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires’ disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insight into how and why the bacterium targets translation elongation remains poorly defined. We recently discovered that the Legionella effector SidI functions unconventionally as a transfer RNA (tRNA) mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-EM structure of SidI revealed an N-terminal domain with an ‘inverted-L’ shape and surface charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase B fold, thereby licensing this effector to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a similar potency as ricin, one of the most powerful toxins known. In Legionella infected cells, the translational pausing activated by SidI uniquely elicits a stress response signature reminiscent of the ribotoxic stress response that is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus, and orchestrates the transcription of stress-inducible genes that promote cell death. Taken together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signaling pathway that regulates cell fate.

The interplay between viruses and host cells is extremely complex, as is the resulting disease dynamics. In the cytoplasm of host cells, (+) ss viral RNAs interact with numerous RNA-binding proteins (RBPs).
Work from our laboratory has shown that HuR, an RBP with multiple functions in RNA processing and translation, relocalizes from the nucleus to the cytoplasm upon Hepatitis C virus (HCV) infection. We have shown that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1.
In parallel, we have shown that HuR binds to SARS-CoV-2 5’UTR. The knock-down and knock-out of HuR reduced viral RNA levels and viral titres. Using an antisense strategy, we were able to reduce the viral RNA level in wildtype cells but not in HuR-knockout cells. Interestingly, results suggest HuR supports SARS-CoV-2 life by promoting differential translational reprogramming of both genomic and subgenomic RNAs.
Taken together, we demonstrate important roles of an RNA binding protein HuR, in two RNA viruses, HCV and SARS-CoV-2, and explored different ways to target it for tackling virus infections.

TBA

Cellular phenotype is largely defined by its transcriptional state, which is in turn governed by transcriptional variability of individual genes and the gene regulatory network. The overall phenotype of a tissue or an organ is best characterized by the composition of various cell types and their respective transcriptional states. In the first part of the presentation, we will discuss a few projects that explore cellular states in healthy and tumor tissues associated with clinical outcome. In the second part, we will discuss deep learning approach to functionally characterize non-coding mutations and polymorphisms and applications to understanding human brain evolution and varying cancer risks across populations.

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Up-Frameshift Suppressor 3 Homolog B (UPF3B) is a nucleocytoplasmic shuttling protein central to the classical nonsense-mediated mRNA decay (NMD) pathway. While UPF3B interacts with spliced mRNAs and promotes their cytoplasmic surveillance, its precise role in NMD remains debated. Some studies suggest partial dispensability, whereas others demonstrate that depletion of UPF3B alongside its paralog UPF3A severely impairs NMD. The clinical importance of UPF3B is underscored by patient-derived sequencing data linking its loss-of-function mutations to neurodevelopmental disorders. These findings highlight the need for mechanistic insights into UPF3B’s contribution to NMD and gene regulation.

To address this, we generated mammalian UPF3B knockout (KO) cell lines using CRISPR/Cas9. Following stable Cas9 induction and sgRNA-directed editing, two UPF3B-KO clones were established with distinct deletions (55-bp and 5-bp). NMD activity was assessed using canonical substrates, including the PTC39 β-globin reporter, alongside transcriptomic profiling via RNA-Seq (STAR, Salmon, DESeq2) and isoform switching analyses (IsoformSwitchAnalyzeR).

UPF3B-KO clones displayed robust NMD impairment, evidenced by upregulation of classical NMD substrates and activation of NMD autoregulatory feedback. Differential expression analysis revealed significant induction of known NMD targets such as GADD45G, GPX1, and SELENOP. Transcript-level analysis identified 165 NMD-regulated and 153 novel PTC+ transcripts specifically stabilized in UPF3B-KO cells. Furthermore, isoform switching analyses uncovered increased expression of NMD-sensitive splice isoforms, including SAT1 and HNRNPA2B1, implicating UPF3B in splicing-associated quality control.

In summary, complete loss of UPF3B is sufficient to compromise NMD, alter transcriptome-wide gene expression, and promote the stabilization of unproductive splice variants. Our findings underscore UPF3B’s pivotal role in coupling alternative splicing with NMD, preventing aberrant isoform accumulation, and maintaining mRNA homeostasis.

The interior of a cell is a very crowded environment. How do cells manage to accumulate all components of a molecular process at the same time in the same place? Long nucleotide sequences can provide platforms for the assembly of proteins and RNAs that carry out a variety of activities. In this presentation I will first discuss efforts to map the functions of 3' untraslated regions of mRNAs and then approaches to uncover elements in non-coding regions of RNAs that regulate processes such as translation. 

Antibiotic resistance is a global pandemic that has emerged as a silent killer. Bacteria have harnessed several mechanisms to evade the effect of antibiotics with drug target modification being a highly efficient strategy utilized by pathogenic systems to render themselves resistant to antibiotics. The ribosome owing to its integral role as the protein synthesis machinery of the cell is a prime target for several antibiotics. Here, we unravel the mechanism of post-transcriptional ribosomal methylation which renders the macrolide lincosamide and streptogramin B ((MLSB) class of antibiotics ineffective. The enzyme Erythromycin-resistance methyltransferases (Erms),1 exclusively harboured by several multi-drug resistant (MDR) pathogens can site specifically methylate a ribosomal base (A2058, E.coli numbering) in the nascent peptide exit tunnel of the 50S ribosomal subunit which then renders the MDRs resistant to MLSB class of drugs. Interestingly, we show that Erm is an opportunistic enzyme that exclusively targets ribosomal precursors. Using Cryogenic Electron Microscopy (Cryo-EM) we have trapped the Erm-precursor complex and showed how in a complex environment, during ribosomal biogenesis, Erm can methylate its substrate selectively. Moreover, corroborating single molecule FRET measurements were performed to understand the dynamic nature of these interactions and decipher states that the enzyme charters to achieve catalysis. Our work dwells into the unique dual base flipping mechanism employed by Erms to achieve catalysis and its evolutionary implications in the design of these enzymes that induce resistance in pathogenic strains.3 Furthermore, the findings help in the identification of allosteric sites distal from the catalytic site of Erm which can serve as druggable targets. Subsequently, we have ongoing efforts towards AI-based drug design to specifically target Erm-based resistance thereby, facilitating ways of reversal of resistance. Overall, we draw a holistic picture of Erm’s action and delineate methods of curbing its pathogenic function.
References:
1. Singh et al., ACS Chem. Biol. 2022 Apr; 17(4):829-839.
2. Bhujbalrao R, and Anand R, J. Am. Chem. Soc. 2019 Jan; 141(4):1425-1429.
3. Bhujbalrao R, et al., J. Biol. Chem. 2022 Aug; 298(8):102208.

Ataxin-2 is a translational control molecule mutated in spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS). While intrinsically disordered domains (IDRs) of Ataxin-2 (Atx2) facilitate mRNP condensation into granules, how IDRs work with structured domains to enable positive and negative regulation of target mRNAs remains unclear. Using TRIBE (Targets of RNA-Binding Proteins Identified by Editing) technology, we identified an extensive dataset of Atx2-target mRNAs in the Drosophila brain. Ataxin-2 interactions with AU-rich elements in 3’UTRs modulate stability/ turnover of a large fraction of these target mRNAs. Further genomic and cell biological analyses of Atx2 domain-deletions demonstrate that Atx2: (a) interacts closely with target mRNAs within mRNP granules; (b) contains distinct protein domains that drive or oppose RNP-granule assembly; and (c) has additional essential roles outside of mRNP granules. These findings increase understanding of neuronal translational control mechanisms and inform strategies for Ataxin-2-based interventions under development for neurodegenerative disease.

TBA

TBA

Ribosome biogenesis is a complex process requiring rRNA and several proteins. Ribosomal RNA processing factor 2 (RPF2) is known to be involved in the processing of 25S rRNA and 5S rRNA. We report the role of RPF2 in plants in growth, development and drought stress response. RPF2 interact with plant-specific SOC1, NAC1 transcription factors and with RPL10, and plays a role in the regulation of flowering and root development. The RPF2 binds to promoters of flowering and root-associated genes. Overexpression of RPF2 in Arabidopsis and N.benthamiana showed a robust phenotype, higher trichomes and early flowering, and the mutants or RNAi lines showed the opposite phenotype. RPF2-OE plants are insensitive to ABA, and mutants showed higher water loss. The RPF2-OE plants are resistant to drought stress compared to wild type and RNAi lines.

The ongoing evolutionary arms race between bacteria and their predatory phages gives rise to the evolution of different anti-phage defense systems. Among these, CRISPR-Cas represents an RNA-mediated adaptive and heritable immune system that protects the bacteria and archaea against the invasion of mobile genetic elements such as phages and plasmids. Exploitation of this immune system gave rise to a widely acclaimed and versatile genome editing technology referred as CRISPR/Cas9 and several variants thereof. In this talk, I will emphasize the centrality of RNA in orchestrating the defense response. The defense mechanism proceeds via three distinct stages: (i) acquisition of immunological memory from the foreign genetic elements and storing it in the genome, (ii) retrieving this immunological memory in the form of RNA and (iii) using this RNA as a guide for target recognition and cleavage. This talk will introduce CRISPR-Cas as a prokaryote-specific defense response and highlight the molecular mechanism that underlie the fascinating functional diversity. Finally, I will provide a compelling narrative how mechanistic understanding of this defense system was instrumental for repurposing them as precision genetic scissors.

Untranslated RNA (UTR) at the 3’-end of a messenger RNA (mRNA) plays an essential role in gene expression. It is marked by addition of a long poly(A)-tail that occurs in two concerted steps - endonucleolytic cleavage followed by polyadenylation. Poly(A) polymerase (PAP) carries out polyadenylation in a cleavage and polyadenylation (CPA) complex associated with >85 protein components. Canonical PAPα/γ is the primary PAP for mRNA polyadenylation in the nucleus. We have identified a variant non-canonical PAP, Star-PAP that selects mRNAs for polyadenylation. Unlike PAPα, Star-PAP targets do not require certain canonical cis-elements such as the U-rich downstream sequence and instead harbor an -AUA- motif sandwiched in a GC rich region for Star-PAP binding. In addition, they are dispensable of canonical CPA factors CstF-64 or WDR33 that recognize the polyadenylation site, and instead require additional coregulator protein RBM10 to assemble the CPA complex. This specificity of target mRNA selection is in turn modulated by kinases including casein kinase, protein kinase C, or phosphatidyl inositol kinase, PIPKI affecting Star-PAP-RBM10 nexus to regulates key cellular processes and disease pathology.

Interestingly, over 70% of human genes have multiple PA-sites at the 3′-UTR that are alternately used (alternative polyadenylation, APA) generating more than one mRNA isoform with different UTR lengths. Changes in the UTR length alter protein expression, and/or affect protein function. We have shown that Star-PAP regulates APA genome wide of genes particularly involved in cardiovascular diseases such as hypertrophy and heart failure. Inherent downregulation of Star-PAP or RBM10 during cardiac hypertrophy results in the PA-site shift altering overall hypertrophy gene program. In addition to the polyadenylation step, cleavage step is also critical in gene control during cardiac remodeling in hypertrophy. We have shown a regulated cleavage imprecision resulting cleavage site (CS) heterogeneity (CSH) centring around a primary CS versus several subsidiary CS affecting gene expression. We discovered an inverse relationship between CSH and antioxidant gene expression on hypertrophic induction. A decrease in the CSH and an increase in the primary CS usage induces antioxidant gene expression. Hypertrophic stimulus stimulates oxidative stress, yet the antioxidant response progressively goes down with hypertrophic progression. This is mediated by compromised CSH from Star-PAP down regulation underscoring the critical role of the two steps of 3’-end processing in the gene regulation.

Condensate biology has been linked to several neurodegenerative diseases. This will be a high level overview of the current state of the literature.

I will discuss our investigations of "biomolecular condensates" using C. elegans as our experimental model system.

Following the discovery that the RNA-binding proteome is far larger than previously anticipated (Castello et al., 2012), riboregulation, the direct control of protein function by RNA, has begun to emerge as a new paradigm of biological control (Horos et al., 2019; Huppertz et al., 2022; Chatterjee et al., 2024; Hentze et al., 2025). We are beginning to understand molecular mechanisms of riboregulation, and I will discuss their implications for cell biology, metabolism and disease mechanisms as well as the new therapeutic opportunities. I will also share very recent unpublished data that add a new dimension to the concept of riboregulation…

Castello, A., B. Fischer, K. Schuschke, R. Horos, B.M. Beckmann, C. Strein, N.E. Davey, D.T. Humphreys, T. Preiss, L.M. Steinmetz, J. Krijgsveld and M.W. Hentze. Insights into RNA biology from an atlas of mammalian mRNA-
binding proteins. Cell 149, 1393-1406, 2012.

Hentze, M.W., P. Sommerkamp, V. Ravi and F. Gebauer, Rethinking RNA-binding proteins: riboregulation challenges prevailing views. Cell, in press, 2025.

Horos, R., M. Büscher, R. Kleinendorst, A.-M. Alleaume, A.K. Tarafder, T. Schwarzl, D. Dziuba, C. Tischer, E.M. Zielonka, A. Adak, A. Castello, W. Huber, C. Sachse and M.W. Hentze, The small non-coding vault RNA1-1 acts
as a riboregulator of autophagy. Cell 176, 1054-1067, 2019.

Huppertz, I., J.I. Perez-Perri, P. Mantas, T. Sekaran, T. Schwarzl, F. Russo, D. Ferring-Appel, L. Dimitrova-Paternoga, E. Kafkia, J. Hennig, P.A. Neveu, K. Patil and M.W. Hentze. Riboregulation of Enolase 1 Activity Controls
Glycolysis and Embryonic Stem Cell Differentiation. Mol. Cell 82, 2666-2680, 2022.

Chatterjee, A., M. Noble, T. Sekaran, V. Ravi, D. Ferring-Appel, T. Schwarzl, R. Rampelt and M.W. Hentze. RNA promotes mitochondrial import of F1-ATP synthase subunit alpha (ATP5A1). doi: https://doi.org/10.1101/2024.08.19.608659.

Long non-coding RNAs (lncRNA) is emerging as a key regulatory RNA in the brain. The activity of lncRNAs in neurons have been majorly limited to the nucleus and our understanding of their functions in subcellular space remains elusive. We have used the genome-wide transcriptomic profiling of total RNA isolated from the synaptic compartment and identified synapse-enriched lncRNAs from the mouse hippocampus. Among these transcripts, we find a synapse-centric role for a novel lncRNA, SynLAMP (Synapse-enriched LncRNA Associated with Memory and Plasticity). SynLAMP is specifically transported to the synaptic compartment upon contextual fear conditioning (CFC). We observed that CFC triggers interaction between SynLAMP and translation repressor FUS. The knockdown of SynLAMP prevents activity-induced dendritic translation. Our data suggests that SynLAMP functions as an activity-regulated molecular decoy to sequester translation repressor FUS. We find that the knockdown of SynLAMP partially occludes the fear memory. The decoy function of synaptic lncRNAs was further favoured by our findings on Cerox1 (Cytoplasmic endogenous regulator of oxidative phosphorylation 1), a synapse-enriched lncRNA that acts as a competitive decoy for miRNAs. We observed that Cerox1 levels decrease following sleep loss, a condition known to impair memory. Sleep deprivation or Cerox1 knockdown led to reduced mitochondrial Electron Transport Chain (ETC) activity and diminished ATP production, whereas overexpression of Cerox1 was sufficient to restore ETC function and ATP levels, even under conditions of sleep deprivation. This regulation is mediated by Cerox1’s ability to sequester miRNAs that target both Cerox1 and essential subunits of ETC complex I, thereby sustaining their expression in a sleep-dependent manner. Importantly, overexpression of Cerox1 alleviated memory deficits caused by sleep loss, while a Cerox1 variant lacking miRNA-binding sites failed to produce such effects. Collectively, these findings uncover a previously unrecognized mechanism of memory regulation mediated by the decoy activity of synapse-enriched lncRNAs.

Ribosome collisions activate the ribotoxic stress response (RSR) mediated by the MAP3K ZAKα (ZAK), which in turn regulates downstream phosphorylation of the MAPKs JNK and p38 and cell fate consequences. Previous in vivo studies have shown that ZAK associates with ribosomes and that this interaction likely occurs through the highly negatively charged C-terminal ribosome binding region. Upon widespread ribosome collisions, ZAK becomes phosphorylated and is released from ribosomes. Despite its central role in cell fate decisions during cellular stress, a structural understanding of the ZAK-ribosome interaction and how this interaction leads to ZAK-activation has remained elusive. Here, we combine biochemistry with structural studies to dissect ZAK interactions with ribosomes in various states both in vivo and in vitro. We identify regions of ZAK necessary for binding to ribosomes under untreated conditions. We discover how interactions between ZAK and the collision-interface mediate ZAK activation. Further, we provide insight into how this process is negatively regulated to prevent constitutive ZAK activation. Collectively, we show that ZAK directly binds at the collided ribosome interface giving molecular insight into kinase regulation at the ribosome.

RNA undergoes a variety of chemical modifications after transcription, which are essential for RNA maturation and function. Over 150 RNA modifications have been identified to date, yet only about 16% of them are conserved across all three domains of life—eukaryotes, archaea, and bacteria. The vast majority of RNA modifications are domain- or species-specific, suggesting that RNA modifications have been independently acquired and fixed during evolution to meet diverse environmental challenges. We are engaged in a project to identify novel RNA modifications from various sources, and have reported 18 modifications so far. Taking advantage of mass spec analysis of RNA modifications, we identified more than 50 genes responsible for tRNA modifications, rRNA modifications as well as mRNA modification. Ribosomal RNAs (rRNAs) contain a wide variety of post-transcriptional modifications which play critical roles in ribosome assembly and function. Recently we discovered two stereoselective rRNA methylations in the peptidyl-transferase center (PTC) of 50S ribosomal subunit in Escherichia coli cultured under anaerobic conditions. We also identified the rlmX gene which encodes a cobalamin-dependent radical SAM methyltransferase responsible for these methylations. Double knockout strain of rlmX and rlhA (responsible for ho5C2501) exhibited anaerobic growth reduction. Biochemical studies showed that protein synthesis and peptide bond formation were promoted by these rRNA modifications. Cryogenic electron microscopy (cryo-EM) structure of E. coli 70S ribosome indicated that these hypoxia-induced rRNA modifications stabilize the P-site and the PTC. These findings demonstrated that ribosomes are activated by the hypoxia-induced rRNA modifications to enhance translational capability, thereby surviving in anaerobic conditions. The physiological importance of RNA modification is highlighted by human diseases caused by aberrant RNA modification. We previously reported a severe reduction in the frequency of tRNA modifications in mitochondrial disease patients, like MELAS and MERRF. These findings provided the first evidence of RNA modification disorder. We call “RNA modopathy” as a new category of human diseases. Recently, we successfully introduced tRNA modifications into mutant tRNAs by expressing tRNA-modifying enzymes in the MELAS patient cells. In this presentation, I am gonna talk on our efforts toward future gene therapy.

RNA undergoes a variety of chemical modifications after transcription, which are essential for RNA maturation and function. Over 150 RNA modifications have been identified to date, yet only about 16% of them are conserved across all three domains of life—eukaryotes, archaea, and bacteria. The vast majority of RNA modifications are domain- or species-specific, suggesting that RNA modifications have been independently acquired and fixed during evolution to meet diverse environmental challenges. We are engaged in a project to identify novel RNA modifications from various sources, and have reported 18 modifications so far. Taking advantage of mass spec analysis of RNA modifications, we identified more than 50 genes responsible for tRNA modifications, rRNA modifications as well as mRNA modification.
Ribosomal RNAs (rRNAs) contain a wide variety of post-transcriptional modifications which play critical roles in ribosome assembly and function. Recently we discovered two stereoselective rRNA methylations in the peptidyl-transferase center (PTC) of 50S ribosomal subunit in Escherichia coli cultured under anaerobic conditions. We also identified the rlmX gene which encodes a cobalamin-dependent radical SAM methyltransferase responsible for these methylations. Double knockout strain of rlmX and rlhA (responsible for ho5C2501) exhibited anaerobic growth reduction. Biochemical studies showed that protein synthesis and peptide bond formation were promoted by these rRNA modifications. Cryogenic electron microscopy (cryo-EM) structure of E. coli 70S ribosome indicated that these hypoxia-induced rRNA modifications stabilize the P-site and the PTC. These findings demonstrated that ribosomes are activated by the hypoxia-induced rRNA modifications to enhance translational capability, thereby surviving in anaerobic conditions.
The physiological importance of RNA modification is highlighted by human diseases caused by aberrant RNA modification. We previously reported a severe reduction in the frequency of tRNA modifications in mitochondrial disease patients, like MELAS and MERRF. These findings provided the first evidence of RNA modification disorder. We call “RNA modopathy” as a new category of human diseases. Recently, we successfully introduced tRNA modifications into mutant tRNAs by expressing tRNA-modifying enzymes in the MELAS patient cells. In this presentation, I am gonna talk on our efforts toward future gene therapy.

The rate and accuracy of protein synthesis (translation) is an important determinant of bacterial growth. Ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and tRNA-modifying enzymes (MEs) are among the key components of the translation repertoire. In rapidly growing bacteria, these components are often present in multiple gene copies (e.g. tRNAs) and exhibit functional backups (e.g. tRNAs and tRNA MEs). However, the costs and consequences of this redundancy are not fully understood. Here, we conducted experimental evolution on 17 E. coli ancestral strains with targeted deletions of distinct translation components and varying extent of translational redundancy. The adaptation strategies observed across 102 independent adaptation lines ranged from phenotypic modulations to point mutations and gene duplications. Strains with large and redundant ancestral translational repertoires potentially adapted via upregulation of the backup components and showed minimal genomic changes. In contrast, strains with a compromised translational repertoire adapted via duplication or overexpression of backup genes. However, the strains with compromised translation repertoires —and no gene-copy backups— often failed to adapt or declined in fitness. Overall, our results unravel how the redundancy of translational repertoire influences bacterial growth and adaptation.

small RNAs are among the main drivers of epigenetic gene regulation. There are three main categories of small RNAs: microRNAs, piRNAs, and endogenous siRNAs. These various small RNA pathways are conserved across most eukaryotes. I will talk about the role of small RNAs in the inheritance of memory of stress using the nematode model, followed by our findings on the role of microRNAs in host-pathogen interaction using the SARS-CoV-2 model. We sequenced small RNAs from SARS-CoV-2-infected cells and identified a miRNA derived from a recently evolved region of the viral genome that targets the 3′UTR of interferon-stimulated genes and represses their expression, thus aiding in host immune evasion. Lastly, I will highlight our group’s new ventures to understand the role of small RNAs in host-parasite interaction using the eukaryotic parasite Toxoplasma as a model.

Compartmentalization plays a crucial role in orchestrating complex biochemical reactions within cells through the presence of both large membrane-bound organelles and smaller, dynamic nucleic acid-protein condensates. In higher eukaryotic cells, numerous RNA-binding proteins possess extensive disordered domains, rendering them susceptible to phase separation on interaction with RNA and forming dynamic liquid-like condensates. Many of these RBPs aggregate in ageing-associated neurodegenerative conditions like ALS and FTD. Our previous work has demonstrated that high nuclear RNA concentration keep aggregation prone RBPs in soluble state. Our studies have shown that global RNA metabolism in neurodegeneration and ageing conditions control the phase behavior and properties of the RNA-RBP condensates mainly stress granules.

TBA

 The process of translation initiation from an mRNA uses a special tRNA called initiator tRNA (tRNA^fMet or i-tRNA). The i-tRNA is aminoacylated with methionine and then formylated with N¹⁰-formyl-tetrahydrofolate (N¹⁰-fTHF). Both methionine and N¹⁰-fTHF are produced via one-carbon metabolism providing a metabolic regulation of mRNA translation. The fidelity of translation initiation by i-tRNA is attributed to the structural features found in its acceptor-, and anticodon-, stems. The extent of i-tRNA formylation regulates its participation exclusively at the step of initiation or also at the step of elongation. Further, the pioneering round of initiation by i-tRNA triggers the final stages of ribosome maturation. In our recent work, use of an anticonvulsant drug, lamotrigine, has provided important insights into the mechanism of the role of IF2 in the process of ribosome maturation.