RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)–GMP, RNA 3'-phosphate, and RNA3'pp5'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii

tRNAs undergo an extensive maturation process including post-transcriptional modifications that influence secondary and tertiary interactions. Precursor and mature tRNAs lacking key modifications are often recognized as aberrant and subsequently targeted for decay, illustrating the importance of modifications in promoting structural integrity. tRNAs also rely on tRNA chaperones to promote the folding

Fluorescent reporters have been widely used in modern biology as a powerful tool in cell lineage tracing during development and in studying the pathogenesis of diseases. RNAscope is a recently developed RNA in situ hybridization method with high specificity and sensitivity. Combined application of these two techniques on skeletal tissue is technically challenging and has not been explored; the reporter

In vivo RNA structure analysis has become a powerful tool in molecular biology, largely due to the coupling of an increasingly diverse set of chemical approaches with high-throughput sequencing. This has resulted in a transition from single target to transcriptome-wide approaches. However, these methods require sequencing depths that preclude studying low abundance targets, which are not sufficiently

DNA sequencing is the current key technology for historic or ancient biological samples and has led to many exciting discoveries in the field of paleogenomics. However, functional insights into tissue identity, cellular composition, or gene regulation cannot be gained from DNA. Recent analyses have shown that, under favorable conditions, RNA can also be sequenced from ancient samples, enabling studies

The stability of mRNAs is fundamental to determining expression level and dynamics. Nonetheless, current approaches for measuring transcript half-lives (e.g., transcription shutoff) are generally toxic or technically complex. Here we describe an alternative strategy for targeted measurements of endogenous mRNA stability that is simple, inexpensive, and nontoxic. Cells are first metabolically labeled

In order to survive to the exposure of DNA damaging agents, cells activate a complex response that coordinates the cellular metabolism, cell cycle progression, and DNA repair. Among many other events, recent evidence has described global changes in mRNA splicing in cells treated with genotoxic agents. Here, we explore further this DNA damage-dependent alternative splicing. Indeed, we show that both

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNA-binding proteins coordinates temporal and spatial translation

The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (Pol) II through gene body nucleosomes during elongation. In the budding yeast, ∼280 noncoding RNA genes highly transcribed in vivo by Pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate

We have recently reported on an experimental model of mitochondrial mistranslation conferred by amino acid exchange V338Y in mitochondrial ribosomal protein MrpS5. Here we used a combination of RNA-seq and metabolic profiling of homozygous transgenic Mrps5V338Y/V338Y mice to analyze the changes associated with the V338Y mutation in postmitotic skeletal muscle. Metabolome analysis demonstrated enhanced

Proper evaluation of the ionic structure of biomolecular systems through X-ray and cryo-EM techniques remains challenging but is essential for advancing our understanding of the underlying structure/activity/solvent relationships. However, numerous studies overestimate the number of Mg2+ in deposited structures due to assignment errors finding their origin in improper consideration of stereochemical

SARS-CoV-2 coronavirus is responsible for the Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into nonstructural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. Despite the presence of NSP1

The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol2) consists of tandem repeats of a consensus heptapeptide Y1 S2 P3 T4 S5 P6 S7 . The CTD recruits numerous proteins that drive or regulate gene expression. The trafficking of CTD-interacting proteins is orchestrated by remodeling CTD primary structure via Ser/Thr/Tyr phosphorylation and proline cis-trans isomerization, which collectively inscribe

Interleukin 7 receptor α-chain is crucial for the development and maintenance of T cells and is genetically associated with autoimmune disorders including multiple sclerosis (MS), a demyelinating disease of the CNS. Exon 6 of IL7R encodes for the transmembrane domain of the receptor and is regulated by alternative splicing: inclusion or skipping of IL7R exon 6 results in membrane-bound or soluble IL7R

Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how non-essential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a non-essential yeast splicing

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2'-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. Because

Non-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain ​E. coli ​sRNAs with proteins Hfq and ProQ. Despite the promise of this system

Ribosomes are the macromolecular machines at the heart of protein synthesis, however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome and this binding

G-quadruplexes (G4s) are four-stranded nucleic acid structures that arise from the stacking of G-quartets, cyclic arrangements of four guanines engaged in Hoogsteen base pairing. Until recently, most RNA G4 structures were thought to conform to a sequence pattern in which guanines stacking within the G4 would also be contiguous in sequence (e.g., four successive guanine trinucleotide tracts separated

Ribosomal RNA (rRNA) carries extensive 2’-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2’-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner.

Correct cellular localization is essential for the function of many eukaryotic proteins and hence cell physiology. Here, we present a synthetic genetic device that allows the control of nuclear and cytosolic localization based on controlled alternative splicing in human cells. The device is based on the fact that an alternative 3′ splice site is located within a TetR aptamer that in turn is positioned

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem–loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function

Stress granules (SGs) are stress-induced RNA–protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple

Cold-inducible RNA binding protein (CIRBP) is a stress-responsive protein that promotes cancer development and inflammation. Critical to most CIRBP functions is its capacity to bind and posttranscriptionally modulate mRNA. However, a transcriptome-wide analysis of CIRBP mRNA targets in cancer has not yet been performed. Here, we use an ex vivo breast cancer model to identify CIRBP targets and mechanisms

Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5′ nucleobase of the guide strand affects the

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules

A key approach for improving siRNA efficacy is chemical modifications. Through an in silico screening of modifications at the 5′-end nucleobase of the guide strand, an adenine-derived compound called 6-(3-(2-carboxyethyl)phenyl)-purine (6-mCEPh-purine) was identified to improve the RNAi activity in cultured human cells and in vivo mouse models. Nevertheless, it remains unclear how this chemical modification

The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multibasic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences

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tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological process such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration suggesting a possible role for these small RNAs in the regulation

The 3’ to 5’ exonuclease Pop2p (Caf1p) is part of the CCR4-NOT deadenylation complex that removes poly(A) tails from mRNAs in cells. Pop2p is structurally conserved in eukaryotes, but S. cerevisiae Pop2p harbors non-canonical amino acids in its catalytic center. The enzymatic properties of S. cerevisiae Pop2p are not well defined. Here we characterize the RNA exonuclease activity of recombinant S.

Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required

RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In mRNAs, the deposition

There is increasing interest among cancer researchers in the study of Piwi-interacting RNAs (piRNAs), a group of small RNAs important for maintaining genome stability in the germline. Aberrant expression of piRNAs in cancer could imply an involvement of these regulatory RNAs in neoplastic transformation. On top of that, it could enable early cancer diagnosis based on RNA analysis in liquid biopsies

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq)

Mitochondrial diseases linked to mutations in mitochondrial (mt) tRNA sequences are common. However, the contributions of these tRNA mutations to the development of diseases is mostly unknown. Mutations may affect interactions with (mt)tRNA maturation enzymes or protein synthesis machinery leading to mitochondrial dysfunction. In human mitochondria, in most cases the first step of tRNA processing is

To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor-acceptor pairs. Angle-dependent FRET measurements have proven

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested

A bacterial noncoding RNA motif almost exclusively associated with pnuC genes was uncovered using comparative sequence analysis. Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found “pnuC motif” RNAs might function as aptamers for a

High-throughput RNA sequencing unveiled the complexity of transcriptome and significantly increased the records of long noncoding RNAs (lncRNAs), which were reported to participate in a variety of biological processes. Identification of lncRNAs is a key step in lncRNA analysis, and a bunch of bioinformatics tools have been developed for this purpose in recent years. While these tools allow us to identify

Most mammalian cytoplasmic tRNAs contain ribothymidine (T) and pseudouridine (Ψ) at positions 54 and 55, respectively. However, some tRNAs contain Ψ at both positions. Several Ψ54-containing tRNAs function as primers in retroviral DNA synthesis. The Ψ54 of these tRNAs is produced by PUS10, which can also synthesize Ψ55. Two other enzymes, TRUB1 and TRUB2, can also produce Ψ55. By nearest-neighbor analyses

A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon–anticodon pairing and slippage of the reading frame by −1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3′ untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3′ termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem–loop RNAs. Many

We propose a model for the replication of primordial protocell genomes that builds upon recent advances in the nonenzymatic copying of RNA. We suggest that the original genomes consisted of collections of oligonucleotides beginning and ending at all possible positions on both strands of one or more virtual circular sequences. Replication is driven by feeding with activated monomers and by the activation

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The editors wish to thank the following individuals whose efforts in reviewing papers for RNA in the past year are greatly appreciated.

RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as cotranscriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We hypothesized that stalling the polymerase downstream

Determination of structure of RNA via NMR is complicated in large part by the lack of a precise parameterization linking the observed chemical shifts to the underlying geometric parameters. In contrast to proteins, where numerous high-resolution crystal structures serve as coordinate templates for this mapping, such models are rarely available for smaller oligonucleotides accessible via NMR, or they

Small regulatory RNAs (sRNAs) play an important role for posttranscriptional gene regulation in bacteria. sRNAs recognize their target messenger RNAs (mRNAs) by base-pairing, which is often facilitated by interactions with the bacterial RNA-binding proteins Hfq or ProQ. The FinO/ProQ RNA-binding protein domain was first discovered in the bacterial repressor of conjugation, FinO. Since then, the functional

Termination of protein biosynthesis is an essential step of gene expression, during which a complete functional protein is released from the ribosome. Premature or inefficient termination results in truncated, nonfunctional, or toxic proteins that may cause disease. Indeed, more than 10% of human genetic diseases are caused by nonsense mutations leading to premature termination. Efficient and sensitive

It is only after recent advances in cryo-electron microscopy that it is now possible to describe at high-resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between an “active” and “inactive” conformation. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow–derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3′ side of pyrimidines with a strong preference for U↓A and C↓A

RNA G-quadruplexes fold almost exclusively into parallel-stranded structures and thus display much less structural diversity than their DNA counterparts. However, also among RNA G-quadruplexes peculiar structural elements can be found which are capable of reshaping the physico-chemical properties of the folded structure. A striking example is provided by a uridine tetrad (U-tetrad) placed on the 3′-terminus

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant

The NineTeen Complex (NTC), also known as pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. We show that NTC-subunit Salsa, the Drosophila ortholog

During zebrafish development, an early type of rRNA is gradually replaced by a late type that is substantially different in sequence. We applied RiboMeth-seq to rRNA from developmental stages for profiling of 2′-O-Me, to learn if changes in methylation pattern were a component of the shift. We compiled a catalog of 2′-O-Me sites and cognate box C/D guide RNAs comprising 98 high-confidence sites, including