RNA

During translation initiation, the 40S small ribosomal subunit is recruited to the mRNA 5' cap and scans the 5' untranslated region (UTR) to locate the start codon. While the mechanism of 40S translocation remains elusive, the RNA helicase eIF4A has long been suspected as the primary molecular motor driving 40S scanning. In this study, we utilized GFP reporter mRNAs to investigate the impact of 5' UTR length on translational efficiency. We found that an 8-fold variation in the length of unstructured 5' UTRs did not lead to substantial changes in translation efficiency in wheat germ extract (WGE) and human HEK293T cell lysate. By contrast, the presence of a stable stem-loop in the middle of the 5' UTR significantly reduced cap-dependent translation. These results suggest that mRNA scanning is not rate-limiting when the UTR is devoid of secondary structure. Inhibition of eIF4A by hippuristanol in cell-free protein synthesis systems yielded an equivalent decrease in translation for mRNAs with short and long unstructured 5' UTRs, indicating that eIF4A may be dispensable for 40S scanning. Our data suggest that helicase-independent one-dimensional diffusion may be the primary mechanism enabling 40S movement along the 5' UTR during initiation.

Deadenylation, the enzymatic shortening of the poly(A) tail, is typically the first committed step of mRNA decay. Deadenylation rates span nearly a 1000-fold range between transcripts and are governed by protein-RNA interactions, including those involving cytoplasmic poly(A)-binding protein (PABPC). Previous work shows that PABPC can straddle the junction between the poly(A) tail and the 3' untranslated region (UTR), but whether this conformation influences deadenylation has not been tested. To investigate how straddling influences deadenylation kinetics, we designed a library of tailed RNA substrates and measured both in vitro deadenylation rates in the presence of PABPC and PABPC binding propensity for each substrate. We found that 3' UTR sequences influence deadenylation through two mechanisms. First, structured UTRs are deadenylated more slowly in the absence of PABPC1, an effect that is alleviated by PABPC1. Second, sequences upstream of the poly(A) tail modulate PABPC1 binding propensity, with tighter binding correlating with slower deadenylation. This relationship is abolished with a PABPC1 mutant lacking UTR-binding capacity. Together, these results show that sequences upstream of the poly(A) tail tune PABPC1 binding and deadenylation rates, likely contributing to the range of deadenylation rates observed for cellular mRNAs.

Codon optimality promotes efficient translation and, as recent research has shown, also extends mRNA lifetimes. However, how control is distributed between translation and mRNA degradation remains unclear. We show that this relative impact depends strongly on the measurement approach. Using fluorescent protein reporters can underestimate codon-optimality-dependent increases in translation efficiency. Conversely, analyses based on poly(A)-selected RNA overestimate the impact on translation, because stable transcripts undergoing poly(A) shortening are often inefficiently captured, leading to skewed protein-to-mRNA ratios. This technical bias is not offset by the marginal decline in ribosomal association observed as mRNAs age. Estimates based on total RNA measurements redistribute some of the control attributed to translation to mRNA stability, making the contributions comparable for mRNAs with shorter coding sequences. For longer mRNAs, codon optimality increasingly controls elongation speed, with a greater effect on translation efficiency than on degradation. These insights highlight the importance of measurement strategy for accurately quantifying the determinants of mRNA stability and protein synthesis.

Nearest neighbor parameters are widely used in software for estimating the conformational stability of an RNA sequence folding into a specific structure. Folding stability for RNA with canonical nucleotides A, C, G, and U has been widely studied, but the same is not true for most modified nucleotides. In this work, we present a comprehensive set of nearest neighbor parameters for estimating the folding stability of RNA including pseudouridine in helical or loop contexts. These parameters are derived from 210 optical melting experiments involving helices with pseudouridine-A and pseudouridine-G pairs and with pseudouridine in loop motifs. The experiments include sequences with pseudouridine and U in the same strand, including U-A and U-G pairs, allowing us to consider the folding stability of sequences with both U and pseudouridine. On average, pseudouridine stabilizes RNA folding compared to U in an analogous motif, although this effect is sequence-context dependent. These parameters improve the modeling of folding stability for RNA secondary structures containing pseudouridine. We demonstrate that these parameters successfully model the secondary structure change for Saccharomyces cerevisiae U2 snRNA when two additional inducible pseudouridines are present. These parameters are freely available and incorporated into the RNAstructure software package. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725682v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@e1167aorg.highwire.dtl.DTLVardef@18ac7f0org.highwire.dtl.DTLVardef@4c909eorg.highwire.dtl.DTLVardef@aa8bca_HPS_FORMAT_FIGEXP M_FIG C_FIG

The subcellular localization patterns of RNAs are controlled by regulatory elements contained within them. However, for most localized RNAs, the identities of these elements remain unknown. We had previously identified several localization elements that are necessary and sufficient for robust, kinesin-dependent RNA targeting to microtubule plus ends in a variety of cell types. Yet the characteristics of these elements that are critical for function remained unclear. To address this, we systematically created tens of thousands of mutant localization elements and quantified their ability to regulate subcellular RNA localization in neuronal cells. We found that the minimally active size of these localization elements is large, approximately 200 nucleotides. These elements contain multiple important subsequences, with some being completely intolerant of any changes and others being tolerant to a shuffling of nucleotide order but not to changes in nucleotide composition. Using single molecule microscopy, we verified these findings in primary rat neurons. Together, these results demonstrate that highly active mammalian RNA localization elements are large, complex, and multipartite and lay a foundation for further mechanistic studies of their function.

Fused in Sarcoma (FUS) is an RNA-binding protein associated with neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia. Along with its structured RNA recognition motif, FUS contains two intrinsically disordered regions (IDRs) that play important roles in RNA recognition. However, structural mechanism and dynamics of these IDRs in recognizing RNA remain elusive. We have used molecular dynamics simulations to investigate the structure and dynamics of the two IDRs, flanking the RRM domain of human FUS, in both Apo and U1 snRNA-bound states. Comparison of structural parameters and molecular interactions reveals that RNA binding stabilizes the IDRs and reduces their conformational flexibility through reorganization of intra-protein contacts and correlated movements. RNA binding also causes rearrangement of backbone dihedral angles of the IDRs and limits the formation of secondary structures such as -helices and 310 helices. Interestingly, the two IDRs exhibit distinct modes of RNA recognition. The N-terminal IDR interacts mainly with the nucleotides located near the terminal regions of the stem-loop snRNA. On the other hand, the C-terminal IDR preferentially associates with the central double-stranded region through extensive interactions with the minor groove of the snRNA. Even within IDRs, we identify specific residues that associate with the snRNA more frequently than others by forming persistent hydrogen bonds. Overall, our findings suggest that the flanking disordered regions of FUS are not merely flexible linkers, but actively participate in RNA recognition through distinct interaction mechanisms. These results provide molecular-level insights into the functional roles of IDRs in recognizing stem-loop RNA.

The bacterial three-hybrid (B3H) assay is a powerful genetic tool for detecting interactions between RNA and RNA-binding proteins (RBPs) and assessing the consequences of RBP mutations. This transcription-based system connects the strength of an RNA-protein interaction to the expression of a lacZ reporter gene in Escherichia coli cells. This in vivo approach allows researchers to dissect RNA-protein interactions within a cellular environment, bypassing the need for biochemical purification of RNAs or proteins. This chapter details a three-day protocol for generating quantitative B3H data. Since a significant challenge in B3H assays is RNA misfolding, we describe a recently optimized set of B3H constructs that mitigates this issue by isolating bait RNAs as discrete folding units.

Split Open Reading frames (Split-ORFs) occur in transcripts containing at least two open reading frames, each encoding a part of the same full-length protein. These multiple open reading frames arise from alternatively spliced transcript isoforms. Split-ORFs have been described in the SR protein family of splicing factors, where the resulting protein halves play important autoregulatory roles. Here, we present the Split-ORF pipeline, a computational tool that predicts Split-ORFs from transcripts sequences and identifies regions unique to the predicted Split-ORF products. Using this pipeline, we predicted more than 14,000 Split-ORF transcripts from alternatively spliced human transcripts containing premature termination codons or retained introns. Hundreds of the Split-ORF unique regions show significant Ribo-seq coverage across diverse cell types and diseases. The candidate Split-ORF genes with significant Ribo-seq coverage are enriched for RNA-binding and RNA-processing functions and the majority of them encodes RNA-binding proteins. Together, these results suggest that Split-ORFs are more widespread than previously assumed and are expressed across diverse cellular contexts. This work paves the road for future studies of the Split-ORF candidates, the mechanisms of their biogenesis and their functions within the RNA-binding protein class.

The RNA demethylase FTO erases N6-methyladenosine (m6A) and cap-associated N6,2'-O-dimethyladenosine (m6Am) modifications. However, the molecular basis of its substrate selectivity and the biological effects of m6A versus m6Am demethylation in cells remain poorly understood. Here we report two engineered FTO separation-of-function mutants to selectively demethylate either m6A or m6Am modifications on RNA. While investigating the propensity of FTO active site residues to undergo self-hydroxylation, we found that mutations of FTO residue L203 resulted in impaired m6A demethylation but retained wild-type levels of m6Am demethylation, and that FTO L203A could function as a selective m6Am demethylase. Conversely, building on our recent work that identified conserved aromatic residues on FTO involved in mRNA 5' cap recognition, we found that the FTO H232A/W278A double mutant efficiently demethylates m6A modifications while exhibiting substantially impaired m6Am demethylation, making it a selective m6A demethylase. Together, these complementary FTO variants represent the first set of engineered mutations that shift FTO demethylation selectivity between m6A and m6Am substrates. These tools enable selective enzymatic removal of m6A or m6Am modifications in vitro for sequencing applications, and may facilitate understanding of FTO-mediated m6A versus m6Am demethylation in cellular and disease model systems.

Splicing is governed by interactions between the spliceosome and precursor RNA sequence and structural elements. However, the relative contributions of RNA sequence and structural elements remain unclear. Here, we systematically dissect these determinants using a high-throughput mutagenesis approach with the MAP3K7 intron reporter. The MAP3K7 gene encodes a serine/threonine kinase involved in response to environmental stress. MAP3K7 precursor RNA contains a cryptic 3 splice site that increases in use when the core spliceosomal protein SF3B1 is mutated. SF3B1 mutations are known to promote aberrant splicing and are associated with cancer, particularly the lysine 700 to glutamate mutation (K700E). We designed a pooled library of 249 MAP3K7 mutants targeting branch points, RNA-binding protein motifs, nucleotide composition and predicted structural elements. The impact of these mutants on splicing was measured in the context of normal and SF3B1 K700E expression. RNA structure was assessed in parallel using in vitro high-throughput SHAPE-MAP chemical probing. We found that branchpoint mutations drive the strongest increases in cryptic splice-site use. There is no overall correlation between cryptic splice-site use and structural similarity to the wild-type MAP3K7 RNA. However, mutants within an RNA binding protein hotspot (containing U2AF2, U2AF1, KHSRP and SRSF2 sites) are associated with cryptic splice-site use and structural similarity to wild-type MAP3K7 RNA. These structural changes are associated with increased ensemble diversity. Our results demonstrate that although there are key structured regions within an RNA, there is also extensive variability where divergent RNA structures allow for accurate splicing.

RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the Picornaviridae family, but only a subset of those tested were functional - all are in Enterovirus coxsackiepol. When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.

Long noncoding RNAs (lncRNAs) regulate diverse cellular processes and are frequently implicated in disease, but their functional mechanisms often remain elusive. One such lncRNA, HOTAIR (Hox transcript antisense intergenic RNA), is a [~]2.1 kb mammalian transcript whose overexpression promotes invasion and metastasis in breast cancer. However, the mechanisms by which HOTAIR influences gene regulation in cancer are poorly understood. To approach this problem through a structural lens, we determined the full-length in cellulo secondary structure of HOTAIR using chemical probing in a metastatic breast cancer cell line. The resulting structure shows that HOTAIR adopts a multidomain architecture and has local structural features unique to the cellular context. Comparison between in vitro and in cellulo chemical probing identifies regions of differential accessibility that may indicate context-dependent molecular interactions or folding. Conservation analyses further reveal that HOTAIR is conserved across primates with evidence of structural covariation in specific domains. Together, these results provide a roadmap for future mechanistic studies of structure-function relationships in HOTAIR and its contribution to gene regulation in cancer.

Transposable element-derived long intergenic noncoding RNAs are increasingly recognized as context-dependent regulators of gene expression, but the functional consequences of their ectopic activation in somatic cells remain poorly understood. We previously showed that U7 snRNA represses a subset of LTR12-associated lincRNAs, including lnc-ARRDC4-1 and lnc-ADCYAP1-2, two testis-enriched lincRNAs with minimal expression in somatic cells. Here, we examined the consequences of their increased expression in somatic cells. We showed that overexpression of either lincRNA led to overlapping transcriptomic and proteomic changes, impaired migration, altered adhesion and proliferation, and a [~]50% reduction in protein synthesis. Furthermore, we identified lnc-ARRDC4-1 as an upstream regulator of lnc-ADCYAP1-2 transcription. Downstream of this event, lnc-ADCYAP1-2 interacts with the RNA helicase DHX36, a regulator of G-quadruplex-containing mRNAs. lnc-ADCYAP1-2 activation reduces DHX36 protein levels which is accompanied by decreased protein output from a subset of DHX36 mRNA targets. At the cellular level, these effects correlate with altered cell proliferation, migration, adhesion, and global translation. Our results suggest a lnc-ARRDC4-1: lnc-ADCYAP1-2 : DHX36 regulatory cascade linking de-repression of LTR12-containing lincRNAs to reduced protein synthesis and altered cellular processes in somatic cells.

Processing of the precursor ribosomal RNAs (pre-rRNAs) is a key aspect of ribosomal subunit assembly that is closely coordinated with other maturation events. The ribonucleases that mediate pre-rRNA cleavages require regulation to ensure that their activities are exerted in a timely manner. Post-translational modifications can influence protein functions, and although many human ribosome assembly factors are reported to be post-translationally modified, most of these sites remain unconfirmed and functional insights are lacking. Here, we show that NOB1, the PIN domain endoribonuclease responsible for cleavage of the 3' end of the 18S rRNA, is phosphorylated within an evolutionarily conserved acidic tract that can be modified by casein kinase II in vitro. Association of NOB1 with pre-ribosomes is independent of these phosphorylations, and lack of NOB1 phosphorylation only mildly perturbs the efficiency of SSU maturation events upstream of 3' end cleavage of the 18S rRNA. Interestingly, our analyses of pre-rRNA levels in cells depleted of NOB1 or lacking its catalytic activity revealed not only accumulation of the 18SE precursor of the 18S rRNA, but also altered levels of pre-rRNAs containing 5' external transcribed spacer (ETS) sequences (43S, 26S and 30S). This suggests that lack of NOB1-mediated pre-rRNA cleavage impairs recycling of assembly factors required during early biogenesis steps, leading to altered kinetics of 5' ETS processing. Taken together these data provide new insights into the role of NOB1 during SSU biogenesis and the post-translational regulation of this ribonuclease.

Pseudouridine ({Psi}) is an important post-transcriptional modification of many noncoding RNAs that is under-characterized in microRNA (miRNA) due to historical limitations in pseudouridine mapping methods. {Psi} modification stabilizes RNA duplex structures and could therefore play an important role in miRNA target binding and repression. To investigate the extent to which mammalian miRNAs are modified with {Psi}, we profiled the modification landscape of short (<30 nt) RNA in human cells and mouse tissues using bisulfite sequencing. Our approach was powered to detect small RNA pseudouridylation based on robust detection of known {Psi} positions in tRNA fragments (tRFs), some of which show tissue-specific patterns of modification. In contrast with tRFs, we find that miRNA pseudouridylation is exceedingly rare, with a single modified miRNA (miR-3068-5p) identified in mouse tissues. Pseudouridylated miR-3068-5p diSerentially repressed predicted miRNA targets with less stable miRNA:mRNA pairing modes. This study fills a long-standing gap in transcriptome-wide {Psi} profiling and reveals a new potential function for {Psi} as a modulator of activity of small regulatory RNAs.

Pseudouridine is the most abundant RNA base modification due to its prevalence in tRNA and rRNA, where it serves as a key modulator of structure and function. Yet even in a widely used model organism, the budding yeast Saccharomyces cerevisiae, the positions of all pseudouridines in tRNA have not been completely annotated. Using Nanopore direct RNA sequencing (DRS), an established method for detecting RNA pseudouridylation positions, we sequenced cytosolic tRNA from eight pseudouridine synthase (PUS) knockout S. cerevisiae strains, including deletion strains of Pus1, Pus3, and Pus7. Analysis of these data verified thirty-four existing pseudouridine annotations and predicted eleven previously unannotated pseudouridine sites. Our analysis revealed DRS signal changes at several non-uridine sites with the loss of a PUS, including apparent changes in modification abundances at position 37 upon deletion of Pus3. LC-MS/MS and primer extension assays, however, indicated no change in the abundance of these modifications with the loss of Pus3. Our analysis underscores the need for caution in interpreting DRS-based signal changes, particularly in modification-dense regions. Combining existing modification annotations for the thirty-one isoacceptors in the Modomics database with our dataset that added annotations for the remaining eleven isoacceptors, we created a map of all detected pseudouridines, and the enzymes responsible for their catalysis, across the forty-two S. cerevisiae cytosolic tRNA isoacceptors.

The RNA-binding proteins Musashi1 and Musashi2 (MSI1 and MSI2) regulate stem cell function and tissue plasticity by modulating mRNA translation. While typically known as translational repressors, the MSI1 and MSI2 proteins can also act as context-dependent activators of mRNA translation, although the mechanism of MSI-mediated translational activation are unknown. Here, we identify Embryonic Lethal Abnormal Vision-like (ELAVL) proteins as essential co-regulators of MSI1-dependent translational activation. In Xenopus laevis oocytes, antisense oligonucleotide knockdown of Elavl4 inhibited progesterone-stimulated maturation and blocked polyadenylation and translation of key MSI target mRNAs, including the Mos and Cyclin B5 mRNAs. Exogenous expression of ELAVL4 rescued these defects, confirming its necessity for maturation and cell cycle progression. Mechanistically, we determined that the ELAVL4 C-terminal domain interacts with the N-terminal RNA recognition motifs of MSI1 in an RNA-independent manner. Mass spectrometry and functional assays revealed this interaction is evolutionarily conserved: mouse ELAVL1 interacts with MSI1 in the pituitary, and human ELAVL1 rescues Elavl4-depleted Xenopus oocytes. Furthermore, knockdown of Elavl1 in a mammalian cell line abrogated MSI-dependent translational activation of a pituitary Prop1 3-UTR mRNA reporter. Our results establish a conserved mechanism where ELAVL family members interact with MSI to promote MSI-dependent mRNA translational activation.

Non-coding RNAs play diverse roles in a wide range of cellular processes, with their spatial structure being pivotal to their function. RNA secondary structure is a key determinant of its overall fold. Given the scarcity of experimentally determined RNA 3D structures, understanding secondary structure is vital for discerning RNA function. Currently, there is no universally effective solution for de novo RNA secondary structure prediction. Existing methods are becoming increasingly complex without marked improvements in accuracy and often overlook critical features such as pseudoknots and alternative folds. Here, we introduce SQUARNA, a new approach to de novo RNA secondary structure prediction that is suitable for both individual RNA analysis and large-scale structural searches. SQUARNA revisits the concept of base pair maximization and develops it into a stem maximization idea coupled with the widely used free energy minimization (MFE) framework. SQUARNA can predict alternative structures and handle pseudoknots of arbitrary complexity. Benchmarking shows that SQUARNA outperforms existing methods, including deep learning models, in both single-sequence and alignment-based RNA secondary structure prediction. SQUARNA seamlessly integrates sequence and alignment information with experimental data, such as residue reactivities obtained by chemical probing, as well as other structural restraints, including automated searches for Rfam database templates, G-quadruplex patterns, and protein-binding motifs. SQUARNA is available as a standalone tool at https://github.com/febos/SQUARNA and as a web server at https://larnal.imol.institute.

Organization of mRNAs into clusters has been observed in many cellular contexts, yet the features that govern this process in vivo remain poorly understood. Using super-resolution microscopy, single-mRNA imaging, and genetic perturbations, we investigated how mRNA concentration, the double-stranded RNA-binding protein Staufen (Stau), and intermolecular base-pairing driven by an RNA palindrome influence clustering of oskar (osk) mRNA in Drosophila embryos. We find that these factors collectively optimize osk clustering by promoting its dimerization and subsequent oligomerization. Both processes depend on all three factors, although oligomerization is more sensitive to their perturbation, indicating that the driving force for osk oligomerization is partially distinct from that governing dimerization. Furthermore, expression of Stau nearly doubles the likelihood of osk dimerization whereas disruption of the palindrome reduces it fourfold indicating that the presence of Stau and the palindrome lowers the concentration threshold of osk mRNA required for dimerization. Notably, insertion of the osk palindrome into a reporter mRNA markedly increased its association with the endogenous osk, further supporting the conclusion that the palindrome potently drives intermolecular base pairing. Importantly, this experiment also identified the palindrome as the major contributor to heterotypic clustering between the endogenous osk and the reporter mRNA. Finally, computational analyses identified a subset of early embryonic mRNAs predicted to harbor palindromes similar to those found in osk. Among these, eIF3a mRNA emerged as a candidate whose clustering may likewise be driven by intermolecular base pairing. Together, our findings raise the possibility that mRNA clustering driven by palindrome-mediated intermolecular base pairing may be more widespread than previously appreciated and may represent an important mechanism for controlling mRNA spatial organization during early Drosophila development.

Analysis of RNA-binding proteome data from different bacterial species revealed many cell wall metabolic enzymes cross-linking to RNA in vivo, hinting that these proteins directly bind RNA. Surprisingly, penicillin-binding proteins (PBPs) were also abundantly identified as putative RNA-binding proteins. The cell surface localisation properties of many of these proteins therefore beg the question at what stage of their cellular life cycle these proteins interact with RNA and what the functional significance is. Here, we characterised the RNA-binding activity of PBP2a, the alternative transpeptidase that confers {beta}-lactam resistance in MRSA. Using in vivo RNA-binding assays, we show that PBP2a interacts with hundreds of transcripts without apparent sequence specificity. Computational analyses identified a possible RNA-binding cleft in PBP2a proximal to its active site. Mutation of only two predicted positively charged residues located in this cleft substantially reduced cross-linking in vivo, implying that RNA recognition is largely dictated by RNA backbone interactions. While PBP2a does not regulate RNA steady-state levels, RNA-binding appears important for proper protein function: an RNA-binding deficient mutant exhibits reduced oxacillin resistance. These findings establish PBP2a as an RNA-binding protein in vivo and provide a framework to investigate how this non-canonical interaction may relate to cell wall biogenesis and {beta}-lactam resistance.