RNA

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish 'self' from 'non-self.' ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5...

The potential presence of 5-methylcytosine as a sparse internal modification of mRNA was first raised in 1975 and a first map of the modification was also part of the epitranscriptomics 'big bang' in 2012. Since then, the evidence for its presence in mRNA has firmed up and initial insights have been gained into the molecular function and broader biological relevance of 5-methylcytosine when present in mRNA. Here, we summarise the status quo of the field, outline some of its current challenges and suggest how to address them in future work...

Viral RNA molecules contain multiple layers of regulatory information. This includes features beyond the primary sequence, such as RNA structures and RNA modifications, including N6-methyladenosine (m6A). Many recent studies have identified the presence and location of m6A in viral RNA and have found diverse regulatory roles for this modification during viral infection. However, to date, viral m6A mapping strategies have limitations that prevent a complete understanding of the function of m6A on individual viral RNA molecules...

Over the past decade, N6-methyladenosine (m6A), emerging as a prevalent and dynamically regulated modification across the transcriptome, reversibly installed, removed, and interpreted by specific binding proteins, playing crucial roles in molecular and biological processes. Within this scope, we consolidate recent advancements of m6A research in plant regarding in gene expression regulation, diverse physiologic and pathogenic processes, as well as crop trial implication to guide discussions on challenges associated with and leveraging epitranscriptome editing for crop improvement...

Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long non-coding RNAs have polyA(+) tails that are often used for positive selection, investigations of polyA(-) RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment...

N1-methyladenosine (m1 A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. M1 A is generally located in the T loop of cytosolic tRNA and between acceptor and D stems of mitochondrial tRNAs, it is involved in tertiary interaction that stabilizes tRNA. Human tRNA m1 A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1 A, a PCR method to assess m1 A levels quantitatively in specific tRNAs has been lacking...

The mammalian mitochondrial proteome comprises over 1000 proteins, with the majority translated from nuclear-encoded messenger RNAs (mRNAs). Mounting evidence suggests many of these mRNAs are localized to the outer mitochondrial membrane (OMM) in a pre-or co-translational state. Upon reaching the mitochondrial surface, these mRNAs are locally translated to produce proteins that are co-translationally imported into mitochondria. Here, we summarize various mechanisms cells employ to localize RNAs, including transfer RNAs (tRNAs), to the OMM and recent technological advancements in the field to study these processes...

Despite being predicted to lack coding potential, cytoplasmic long non-coding (lnc)RNAs can associate with ribosomes. However, the landscape and biological relevance of lncRNAs translation remains poorly studied. In yeast, cytoplasmic Xrn1-sensitive lncRNAs (XUTs) are targeted by the Nonsense-Mediated mRNA Decay (NMD), suggesting a translation-dependent degradation process. Here, we report that XUTs are pervasively translated, which impacts their decay. We show that XUTs globally accumulate upon translation elongation inhibition, but not when initial ribosome loading is impaired...

In spliceosome assembly, the 5' splice site is initially recognized by U1snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to maintenance of 5' splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in C. elegans at SNRP-27 M141 promote changes in alternative 5'ss usage...

Genome-derived microRNAs (miRNA or miR) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent works have demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers...

UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions however makes attribution of RNA binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA binding protein (RBP) confidence scoring metric, (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high and low confidence candidate RBPs...

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR or DNA cloning. The oligonucleotide design, however, is a challenge to non-experts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the Rational design of Oligonucleotides, Calculated with Kinetic parameters for Enhanced in vitro Transcription (ROCKET)...

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella...

Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae. The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small-molecule inhibitors to disrupt the promoter function...

RNA caps are deposited at the 5'end of RNA-polymerase II transcripts. This modification regulates several steps of gene expression, in addition to marking transcripts as self to enable the innate immune system to distinguish them from uncapped foreign RNAs, including those derived from viruses. Specialized immune sensors, such as RIG-I and IFITs, trigger antiviral responses upon recognition of uncapped cytoplasmic transcripts. Interestingly, uncapped transcripts can also be produced by mammalian hosts. For instance, 5'-triphosphate RNAs are generated by RNA-polymerase III transcription, including tRNAs, Alu RNAs, or vault RNAs...

Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease (TSEN), followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections...

3' untranslated regions (3'UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBP) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localisation. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3'UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear...

The histone lysine demethylase KDM5B is frequently upregulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with miR-140-3p level in primary human AML cells...

Next generation RNA sequencing allows alternative splicing (AS) quantification with unprecedented resolution, with the relative inclusion of an alternative sequence in transcripts being commonly quantified by the proportion of reads supporting it as percent spliced-in (PSI). However, PSI values do not incorporate information about precision, proportional to the respective AS events' read coverage. Beta distributions are suitable to quantify inclusion levels of alternative sequences, using reads supporting their inclusion and exclusion as surrogates for the two distribution shape parameters...

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