Dyskerin and its homologues are ancient and conserved enzymes that catalyse the most common posttranscriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently – it is the core component of a protein heterotetramer, which associates with RNAs that

Intracellular condensates formed through liquid-liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140 and CHERP form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons

BACKGROUND: Conversion of adenosine to inosine in RNA by ADAR enzymes occurs at thousands of sites in the human transcriptome, and is essential for healthy brain development. This editing process is dysregulated in many neuropsychiatric diseases, but has not yet been investigated at the level of individual neurons. METHODS: We quantified RNA editing sites in full-length capture nuclear transcriptomes

Type I toxin-antitoxin (T1TA) systems constitute a large class of genetic modules with antisense RNA (asRNA)-mediated regulation of gene expression. They are widespread in bacteria and consist of an mRNA coding for a toxic protein and a noncoding asRNA that acts as an antitoxin preventing the synthesis of the toxin by directly basepairing to its cognate mRNA. The co- and post-transcriptional regulation

DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y. While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome

RNA 27: 324–334 (2021)

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration

XRN1 is a highly conserved exoribonuclease which degrades uncapped RNAs in a 5′–3′ direction. Degradation of RNAs by XRN1 is important in many cellular and developmental processes and is relevant to human disease. Studies in D. melanogaster demonstrate that XRN1 can target specific RNAs, which have important consequences for developmental pathways. Osteosarcoma is a malignancy of the bone and accounts

The widespread ykkC-I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the Burkholderia sp. TJI49 ykkC-I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from Dickeya dadantii and Sulfobacillus acidophilus

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8–P4 and L18–P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr)

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that

PIWI-interacting (pi)RNAs are small silencing RNAs that are crucial for the defense against transposable elements in germline tissues of animals. In Aedes aegypti mosquitoes, the piRNA pathway also contributes to gene regulation in somatic tissues, illustrating additional roles for piRNAs and PIWI proteins besides transposon repression. Here, we identify a highly abundant endogenous piRNA (propiR1)

CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3′-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5′–3′ exonuclease activity in the U7 machinery, degrading the downstream cleavage product

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a nuclear long noncoding RNA (lncRNA) that is highly overexpressed in many cancer tissues and plays important roles in tumor progression and metastasis. The MALAT1 primary transcript contains evolutionarily conserved structural elements in its 3′-terminal region: a triple helix forming element called element for nuclear expression

Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification

Although long noncoding RNAs (lncRNAs) are generally expressed at low levels, emerging evidence has revealed that many play important roles in gene regulation by a variety of mechanisms as they engage with proteins. Given that the abundance of proteins often greatly exceeds that of their interacting lncRNAs, quantification of the relative abundance, or even the exact stoichiometry in some cases, within

During pre-mRNA maturation 3p end processing can occur at different polyadenylation sites in the 3 prime untranslated region (3p UTR) to give rise to transcript isoforms that differ in the length of their 3p UTRs. Longer 3p UTRs contain additional cis-regulatory elements that impact the fate of the transcript and/or of the resulting protein. Extensive alternative polyadenylation (APA) has been observed

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth

The expression of bromodomain-containing proteins that regulate chromatin structure and accessibility must be tightly controlled to ensure the appropriate regulation of gene expression. In the yeast S. cerevisiae, Bromodomain Factor 2 (BDF2) expression is extensively regulated post-transcriptionally during stress by RNase III-mediated decay (RMD), which is triggered by cleavage of the BDF2 mRNA in

Stress-induced molecular damage to ribosomes can impact protein synthesis in cells, but cell-based assays do not provide a clear way to distinguish the effects of ribosome damage from stress responses and damage to other parts of the translation machinery. Here we describe a detailed protocol for the separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution

Even though microRNAs have been viewed as promising biomarkers for years, their clinical implementation is still lagging far behind. This is in part due to the lack of RT-qPCR technologies that can differentiate between microRNA isoforms. For example, A-to-I editing of microRNAs through adenosine deaminase acting on RNA (ADAR) enzymes can affect their expression levels and functional roles, but editing

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3′ nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated

Polyadenylated nuclear (PAN) RNA is a long noncoding transcript involved in Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have previously shown that PAN RNA has dynamic secondary structure and protein binding profiles that can be influenced by epitranscriptomic modifications. N6-methyladenosine (m6A) is one of the most abundant

The expression of long noncoding RNAs is highly enriched in the human nervous system. However, the function of neuronal lncRNAs in the cytoplasm and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of human SH-SY5Y cells. We discovered

Sense-antisense mRNA pairs generated by convergent transcription is a way of gene regulation. c-fms gene is closely juxtaposed to the HMGXB3 gene in the opposite orientation, in chromosome 5. The intergenic region (IR) between c-fms and HMGXB3 genes is 162 bp. We found that a small portion (∼4.18%) of HMGXB3 mRNA is transcribed further downstream, including the end of the c-fms gene generating antisense

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia,

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host

Connections between distinct catalytic RNA motifs through networks of mutations that retain catalytic function (neutral networks) were likely central to the evolution of biocatalysis. Despite suggestions that functional RNAs collectively form an interconnected web of neutral networks, little evidence has emerged to demonstrate the existence of such intersecting networks in naturally occurring RNAs

TNRC6 is a scaffolding protein that bridges interactions between small RNAs, argonaute (AGO) protein, and effector proteins to control gene expression. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C. These paralogs have ∼40% amino acid sequence identity and the extent of their unique or redundant functions is unclear. Here, we use knockout cell lines, enhanced crosslinking

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic

In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility

In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified posttranscriptionally. Pseudouridylation and 2’-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based

Pseudouridine (ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes ~7% of the total uridine level. However, ψ constitutes only ~0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by

In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme’s hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multi-component RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference

Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 de-methylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes

A universal property of all rRNAs explored to date is the prevalence of post-transcriptional (‘epitranscriptional’) modifications, which expand the chemical and topological properties of the four standard nucleosides. Are these modifications an inert, constitutive part of the ribosome? Or could they, in part, also regulate the structure or function of the ribosome? In this review we summarize emerging

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed

Serine/Arginine Splicing Factor 10 (SRSF10) is a member of the family of mammalian splicing regulators known as SR proteins. Like several of its SR siblings, the SRSF10 protein is composed of an RNA binding domain (RRM) and of arginine and serine-rich auxiliary domains (RS) that guide interactions with other proteins. The phosphorylation status of SRSF10 is of paramount importance for its activity

During protein synthesis on ribosome, tRNA recognizes its cognate codon of mRNA through base pairing with the anticodon. The 5’-end nucleotide of the anticodon is capable of wobble base pairing, offering a molecular basis for codon degeneracy. The wobble nucleotide is often targeted for post-transcriptional modification, which affects the specificity and fidelity of the decoding process. Flipping-out

Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3′-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction, PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3′-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AngII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins

Ewing sarcoma is driven by fusion proteins containing a low-complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend leukemia virus integration

Noncoding RNAs play a crucial role in various cellular processes in living organisms, and RNA functions heavily depend on molecule structures composed of stems, loops, and various tertiary motifs. Among those, the most frequent are A-minor interactions, which are often involved in the formation of more complex motifs such as kink-turns and pseudoknots. We present a novel classification of A-minors

Enhancer RNAs (eRNA) are noncoding transcripts produced from active enhancers and have potential gene regulatory function. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor generally involved in metabolism, cell cycle inhibition, hematopoiesis, adipogenesis, hepatogenesis, and is associated with tumorigenesis. In this study, we demonstrate that an enhancer-associated long noncoding

Mammalian C-to-U RNA editing was described more than 30 yr ago as a single nucleotide modification in small intestinal Apob RNA, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory

Long-read transcriptome sequencing is designed to sequence full-length RNA molecules and advantageous for identifying alternative splice isoforms; however, in the absence of a reference genome, it is difficult to accurately locate splice sites because of the diversity of patterns of alternative splicing (AS). Based on long-read transcriptome data, we developed a versatile tool, IsoSplitter, to reverse-trace

In prokaryotes, CRISPR-Cas immune systems recognize and cleave foreign nucleic acids to defend against mobile genetic elements (MGEs). Type III CRISPR-Cas complexes also synthesize cyclic oligoadenylate (cOA) second messengers, which activate CRISPR ancillary proteins involved in antiviral defense. In particular, cOA-stimulated nucleases degrade RNA and DNA nonspecifically, which slows MGE replication

Pre-mRNA splicing is carried out by the spliceosome and involves splice site recognition, removal of introns, and ligation of exons. Components of the spliceosome have been shown to interact with the elongating RNA polymerase II (RNAPII), which is thought to allow splicing to occur concurrently with transcription. However, little is known about the regulation and efficiency of cotranscriptional splicing

The function of an RNA is intimately linked to its structure. Many approaches encompassing X-ray crystallography, NMR, structural probing, or in silico predictions have been developed to establish structural models, sometimes with a precision down to atomic resolution. Yet these models still require experimental validation through the preparation and functional assay of mutants, which can rapidly become

The stem cell–specific RNA-binding protein TRIM71/LIN-41 was the first identified target of the prodifferentiation and tumor suppressor miRNA let-7. TRIM71 has essential functions in embryonic development and a proposed oncogenic role in several cancer types, such as hepatocellular carcinoma. Here, we show that TRIM71 regulates let-7 expression and activity via two independent mechanisms. On the one

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, σB, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed