Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2′-OMe, and Ψ were detected. However, since their abundances are low and tools used for

Ribosome footprint profiling is a high-throughput sequencing–based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued as a standalone

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved,

RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as

The majority of mouse and human genes are subject to alternative cleavage and polyadenylation (APA), which most often leads to the expression of two or more alternative length 3′ untranslated region (3′-UTR) mRNA isoforms. In neural tissues, there is enhanced expression of APA isoforms with longer 3′-UTRs on a global scale, but the physiological relevance of these alternative 3′-UTR isoforms is poorly

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1–7 complex initiates mRNA decay, while the nuclear Lsm2–8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes

Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression

Pat1, known as Pdc2 in fission yeast, promotes the activation and assembly of multiple proteins during mRNA decay. After deadenylation, the Pat1/Lsm1–7 complex binds to transcripts containing oligo(A) tails, which can be modified by the addition of several terminal uridine residues. Pat1 enhances Lsm1–7 binding to the 3′ end, but it is unknown how this interaction is influenced by nucleotide composition

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their

Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3′ end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1, which is normally repressed by transcription in cis of a 5′ flanking prt lncRNA. Here we present evidence that de-repression of pho1 by erh1Δ is achieved through precocious 3′-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity

Single-cell RNA sequencing (scRNA-seq) is a recent technology that enables fine-grained discovery of cellular subtypes and specific cell states. Analysis of scRNA-seq data routinely involves machine learning methods, such as feature learning, clustering, and classification, to assist in uncovering novel information from scRNA-seq data. However, current methods are not well suited to deal with the substantial

Human CD4+ T cells are often subdivided into distinct subtypes, including Th1, Th2, Th17, and Treg cells, that are thought to carry out distinct functions in the body. Typically, these T-cell subpopulations are defined by the expression of distinct gene repertoires; however, there is variability between studies regarding the methods used for isolation and the markers used to define each T-cell subtype

In a recently published paper, Huang and coworkers claim that proteins translated in different reading frames from the same mRNA can have similar functions. This conclusion is possibly incorrect due to the possibility that the wild-type protein could still be expressed.

Like most RNA viruses, influenza viruses generate defective viral genomes (DVGs) with large internal deletions during replication. There is accumulating evidence supporting a biological relevance of such DVGs. However, further understanding of the molecular mechanisms that underlie the production and biological activity of DVGs is conditioned upon the sensitivity and accuracy of detection methods,

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

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 catalogue of 2’-O-Me sites and cognate box C/D guide RNAs comprising 98 high-confidence sites,

Spliced leader trans-splicing is essential for the processing and translation of polycistronic RNAs generated by eukaryotic operons. In C. elegans, a specialised spliced leader, SL2, provides the 5’ end for uncapped pre-mRNAs derived from polycistronic RNAs. Studies of other nematodes suggested that SL2-type trans-splicing is a relatively recent innovation, confined to Rhabditina, the clade containing

Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA Editing Substrate binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C). RESC and REH2C stably co-purify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include mis-edited ‘junction’

Molecular analysis of RNA through hybridization with sequence-specific probes is challenging due to the intrinsic ability of RNA molecules to form stable secondary and tertiary structures. To overcome the energy barrier towards the probe-RNA complex formation, the probes are made of artificial nucleotides, which are more expensive than their natural counterparts and may still be inefficient. Here,

Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5’ to 3’ exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details

The Fibro-purF motif is a putative structured noncoding RNA domain that was discovered previously in species of Fibrobacter by employing comparative sequence analysis methods. An updated bioinformatics search yielded a total of only 30 unique-sequence representatives, exclusively found upstream of the purF gene that codes for the enzyme amidophosphoribosyltransferase. This enzyme synthesizes the compound

In vitro, Drosophila melanogaster Dicer-2 (Dcr-2) uses its helicase domain to initiate processing of dsRNA with blunt (BLT) termini, and its Platform•PAZ domain to initiate processing of dsRNA with 3’ overhangs (ovrs). To understand the relationship of these in vitro observations to roles of Dcr-2 in vivo, we compared in vitro effects of two helicase mutations to their impact on production of endogenous

Ribonucleic acids (RNAs) play essential roles in living cells. Many of them fold into defined three-dimensional (3D) structures to perform functions. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have enabled structure determinations of RNA to atomic resolutions. However, most RNA molecules are structurally flexible, limiting the resolution of their structures solved by cryo-EM

MiR-140 is selectively expressed in cartilage. Deletion of the entire miR-140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology, however the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human

RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in disease such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we

Cellular quiescence and cell cycle re-entry regulate vital biological processes such as cellular development and tissue homeostasis, and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs

Viruses have evolved in tandem with the organisms that they infect. Afflictions of the plant and animal kingdoms with viral infections have forced the host organism to evolve new or exploit existing systems to develop the countermeasures needed to offset viral insults. As one example, nonsense-mediated mRNA decay, a cellular quality-control mechanism ensuring the translational fidelity of mRNA transcripts

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex

Nucleic acid aptamers can be chemically modified to enhance function, but modifying previously selected aptamers can have non-trivial structural and functional consequences. We present a reselection strategy to evaluate the impact of several modifications on pre-existing aptamer pools. RNA aptamer libraries with affinity to HIV-1 reverse transcriptase (RT) were re-transcribed with 2′-F, 2′-OMe, or

tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb data base (The Lowe Lab, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for Alanine compared to other tRNA

Chaperone proteins --- the most disordered among all protein groups --- help RNAs fold into their functional structure by destabilizing misfolded configurations or stabilizing the functional ones. But disentangling the mechanism underlying RNA chaperoning is challenging, mostly due to inherent disorder of the chaperones and the transient nature of their interactions with RNA. In particular, it is unclear

Mechanisms underlying the ability of hepatitis C virus (HCV) to establish persistent infections and induce progressive liver disease remain poorly understood. HCV is one of several positive-stranded RNA viruses capable of establishing persistence in their immunocompetent vertebrate hosts, an attribute associated with formation of large scale RNA structure in their genomic RNA. We developed novel methods

Programmed Cell Death 4 (PDCD4) protein is a tumour suppressor that inhibits translation through the mTOR dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons, with loss and gain of function experiments in cortical and dorsal root ganglia primary

The human PUF-family proteins, PUM1 and PUM2, post-transcriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3’ UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic

PHAX (Phosphorylated adaptor for RNA export) promotes nuclear export of short transcripts of RNA polymerase II such as spliceosomal U snRNA precursors, as well as intra-nuclear transport of small nucleolar RNAs (snoRNAs). However, it remains unknown whether PHAX has other critical functions. Here we show that PHAX is required for efficient DNA damage response (DDR) via regulation of phosphorylated

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can

The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a

Native folded and compact intermediate states of RNA typically involve tertiary structures in the presence of divalent ions such as Mg2+ in a background of monovalent ions. In a recent study we have shown how the presence of Mg2+ impacts the transition from partially unfolded to folded states through a “push-pull” mechanism where the ion both favors and disfavors the sampling of specific phosphate-phosphate

The fission yeast Schizosaccharomyces pombe is an excellent model organism for the study of eukaryotic cellular physiology. The organism is genetically tractable and several tools to study the functions of individual genes are available. One such tool is regulatable gene expression and overproduction of proteins. Limitations of currently available overexpression systems include: delay in expression

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, non-functional 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

Staufen1 (STAU1) is an RNA-binding protein (RBP) that interacts with double-stranded RNA structures and has been implicated in regulating different aspects of mRNA metabolism. Previous studies have indicated that STAU1 interacts extensively with RNA structures in coding regions (CDSs) and 3'-untranslated regions (3'UTRs). In particular, duplex structures formed within 3'UTRs by inverted-repeat Alu

In addition to adenosine-to-inosine RNA editing activities, ADAR1 has been shown to have various RNA editing independent activities including modulation of RNAi efficacy. We previously reported that ADAR1 forms a heterodimer complex with DICER and facilitates processing of pre-miRNAs to mature miRNAs. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs)

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA

Queuosine (Q) is a conserved tRNA modification in bacteria and eukaryotes. Eukaryotic Q-tRNA modification occurs through replacing the guanine base with the scavenged metabolite queuine at the wobble position of tRNAs with G34U35N36 anticodon (Tyr, His, Asn, Asp) by the QTRT1/QTRT2 heterodimeric enzyme encoded in the genome. In humans, Q-modification in tRNATyr and tRNAAsp are further glycosylated

Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5’,3’-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems,

PPR proteins are a diverse family of RNA binding factors found in all Eukaryotic lineages. They perform multiple functions in the expression of organellar genes, mostly on the post-transcriptional level. PPR proteins are also significant determinants of evolutionary nucleo-organellar compatibility. Plant PPR proteins recognize their RNA substrates using a simple modular code. No target sequences recognized

During breast cancer metastasis, the developmental process epithelial–mesenchymal transition (EMT) is abnormally activated. Transcriptional regulatory networks controlling EMT are well-studied; however, alternative RNA splicing also plays a critical regulatory role during this process. A comprehensive understanding of alternative splicing (AS) and the RNA binding proteins (RBPs) that regulate it during

We have previously shown that when the uridine of a stop codon (UAA, UAG, or UGA) is pseudouridylated, the ribosome reads through the modified stop codon. However, it is not clear as to whether or not the pseudouridine (Ψ)-mediated readthrough is dependent on the sequence context of mRNA. Here, we use several different approaches and the yeast system to address this question. We show that when a stop

The wide prevalence and regulated expression of long noncoding RNAs (lncRNAs) highlight their functional roles, but the molecular basis for their activities and structure-function relationships remains to be investigated, with few exceptions. Among the relatively few lncRNAs conserved over significant evolutionary distances is the long intergenic noncoding RNA (lincRNA) Cyrano (orthologous to human

In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent

In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary

The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral

Influenza A virus (IAV) utilizes cap-snatching to obtain host capped small RNAs for priming viral mRNA synthesis, generating capped hybrid mRNAs for translation. Previous studies have been focusing on canonical cap-snatching, which occurs at the very 5′ end of viral mRNAs. Here we discovered noncanonical cap-snatching, which generates capped hybrid mRNAs/noncoding RNAs mapped to the region ∼300 nucleotides

DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli)

Tob2, an anti-proliferative protein, promotes deadenylation through recruiting Caf1 deadenylase to the mRNA poly(A) tail by simultaneously interacting with both Caf1 and poly(A)-binding protein (PABP). Previously, we found that changes in Tob2 phosphorylation can alter its PABP-binding ability and deadenylation-promoting function. However, it remained unknown regarding the relevant kinase(s). Moreover

tRNAs constitute the most highly modified class of RNA. Every tRNA contains a unique set of modifications, and Ψ55, m5U54, and m7G46 are frequently found within the elbow of the tRNA structure. Despite the abundance of tRNA modifications, we are only beginning to understand the orchestration of modification enzymes during tRNA maturation. Here, we investigated whether pre-existing modifications impact