scholarly journals Terminal Modification, Sequence, and Length Determine Small RNA Stability in Animals

2020 ◽  
Author(s):  
Ildar Gainetdinov ◽  
Cansu Colpan ◽  
Katharine Cecchini ◽  
Paul Albosta ◽  
Karina Jouravleva ◽  
...  
Keyword(s):  
Small Rna ◽  
Viral Infections ◽  
Rna Stability ◽  
Degradation Pathway ◽  
Decay Rates ◽  
List Type ◽  
Piwi Protein ◽  
Rna Modifications ◽  
Regulate Gene Expression ◽  
Pirna Pathway

ABSTRACTIn animals, piRNAs, siRNAs, and miRNAs silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3′ terminal trimming and 2′-O-methylation. Both trimming and methylation influence piRNA stability. Here, we report that trimming and methylation protect mouse piRNAs from different decay mechanisms. In the absence of 2′-O-methylation, mouse piRNAs with extensive complementarity to long RNAs become unstable. In flies, 2′-O-methylation similarly protects both piRNAs and siRNAs from complementarity-dependent destabilization. Animal miRNAs are unmethylated, and complementarity-dependent destabilization helps explain differences in miRNA decay rates in both mice and flies. In contrast, trimming protects mouse piRNAs from a separate degradation pathway unaffected by target complementarity but sensitive to the 3′ terminal, untrimmed sequence. Because distinct sets of mouse piRNAs are protected by each of these mechanisms, loss of both trimming and 2′-O-methylation causes the piRNA pathway to collapse, demonstrating that these two small RNA modifications collaborate to stabilize piRNAs.Highlights2′-O-methylation protects mouse and fly piRNAs from complementarity-dependent decay2′-O-methylation protects fly siRNAs with extensive complementarity to long RNAsComplementarity to long RNAs predicts the half-life of fly and mouse miRNAsMouse pre-piRNA decay reflects both pre-piRNA sequence and PIWI protein identity

scholarly journals Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

2020 ◽  
Author(s):  
Michael B. Wolfe ◽  
Trista L. Schagat ◽  
Michelle T. Paulsen ◽  
Brian Magnuson ◽  
Mats Ljungman ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Rna Stability ◽  
Decay Rates ◽  
Metabolic Labeling ◽  
Rna Turnover ◽  
Regulate Gene Expression ◽  
Recognition Element ◽  
Rna Targets ◽  
Functional Regulation

AbstractThe 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 labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a ‘rulebook’ of key contextual features that differentiate functional vs. non-functional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

scholarly journals Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

2020 ◽  
Author(s):  
Nadine Körtel ◽  
Cornelia Rücklé ◽  
You Zhou ◽  
Anke Busch ◽  
FX Reymond Sutandy ◽  
...  
Keyword(s):  
Machine Learning ◽  
Rna Stability ◽  
Rna Modification ◽  
List Type ◽  
Rna Modifications ◽  
Uv Crosslinking ◽  
Input Material ◽  
Machine Learning Model ◽  
Eukaryotic Mrnas ◽  
Nucleotide Resolution

AbstractN6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing, such as RNA stability and translation. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites in the transcriptome with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present several experimental and computational innovations, that significantly improve transcriptome-wide detection of m6A sites. Based on the recently developed iCLIP2 protocol, the optimised miCLIP2 results in high-complexity libraries from less input material, which yields a more comprehensive representation of m6A sites. Next, we established a robust computational pipeline to identify true m6A sites from our miCLIP2 data. The analyses are calibrated with data from Mettl3 knockout cells to learn the characteristics of m6A deposition, including a significant number of m6A sites outside of DRACH motifs. In order to make these results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.HighlightsmiCLIP2 produces complex libraries to map m6A RNA modificationsMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sitesMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KOm6A modifications frequently occur outside of DRACH motifs and associates with alternative splicing

scholarly journals Regulation of RNA stability at the 3′ end

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Mallory I. Frederick ◽  
Ilka U. Heinemann
Keyword(s):  
Mrna Decay ◽  
Rna Stability ◽  
Rna Modifications ◽  
Dual Roles ◽  
Cellular Pathways ◽  
Low Levels

AbstractRNA homeostasis is regulated by a multitude of cellular pathways. Although the addition of untemplated adenine residues to the 3′ end of mRNAs has long been known to affect RNA stability, newly developed techniques for 3′-end sequencing of RNAs have revealed various unexpected RNA modifications. Among these, uridylation is most recognized for its role in mRNA decay but is also a key regulator of numerous RNA species, including miRNAs and tRNAs, with dual roles in both stability and maturation of miRNAs. Additionally, low levels of untemplated guanidine and cytidine residues have been observed as parts of more complex tailing patterns.

scholarly journals Functional profiling of COVID-19 respiratory tract microbiomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Niina Haiminen ◽  
Filippo Utro ◽  
Ed Seabolt ◽  
Laxmi Parida
Keyword(s):  
Respiratory Tract ◽  
Carbohydrate Metabolism ◽  
Lower Respiratory Tract ◽  
Viral Infections ◽  
Lavage Fluid ◽  
Degradation Pathway ◽  
Community Acquired Pneumonia ◽  
The Body ◽  
Sequencing Data ◽  
Functional Profiling

AbstractIn response to the ongoing global pandemic, characterizing the molecular-level host interactions of the new coronavirus SARS-CoV-2 responsible for COVID-19 has been at the center of unprecedented scientific focus. However, when the virus enters the body it also interacts with the micro-organisms already inhabiting the host. Understanding the virus-host-microbiome interactions can yield additional insights into the biological processes perturbed by viral invasion. Alterations in the gut microbiome species and metabolites have been noted during respiratory viral infections, possibly impacting the lungs via gut-lung microbiome crosstalk. To better characterize microbial functions in the lower respiratory tract during COVID-19 infection, we carry out a functional analysis of previously published metatranscriptome sequencing data of bronchoalveolar lavage fluid from eight COVID-19 cases, twenty-five community-acquired pneumonia patients, and twenty healthy controls. The functional profiles resulting from comparing the sequences against annotated microbial protein domains clearly separate the cohorts. By examining the associated metabolic pathways, distinguishing functional signatures in COVID-19 respiratory tract microbiomes are identified, including decreased potential for lipid metabolism and glycan biosynthesis and metabolism pathways, and increased potential for carbohydrate metabolism pathways. The results include overlap between previous studies on COVID-19 microbiomes, including decrease in the glycosaminoglycan degradation pathway and increase in carbohydrate metabolism. The results also suggest novel connections to consider, possibly specific to the lower respiratory tract microbiome, calling for further research on microbial functions and host-microbiome interactions during SARS-CoV-2 infection.

scholarly journals An Evolutionarily Conserved piRNA-producing Locus Required for Male Mouse Fertility

2018 ◽  
Author(s):  
Pei-Hsuan Wu ◽  
Yu Fu ◽  
Katharine Cecchini ◽  
Deniz M. Özata ◽  
Amena Arif ◽  
...  
Keyword(s):  
Male Mouse ◽  
Biological Function ◽  
In Utero ◽  
Normal Male ◽  
List Type ◽  
Embryo Viability ◽  
Male Sterile ◽  
Direct Role ◽  
Protein Mutants ◽  
Pirna Pathway

SUMMARYPachytene piRNAs, which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like miRNAs, cleave targets like siRNAs, or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired bypi6−/−fathers show reduced viability in utero. Molecular analyses suggest thatpi6piRNAs repress gene expression by cleaving mRNAs encoding proteins required for sperm function.pi6also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10 as well aspi6itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.HighlightsNormal male mouse fertility and spermiogenesis require piRNAs from thepi6locusSperm capacitation and binding to the zona pellucida of the egg requirepi6piRNAsHeterozygous embryos sired bypi6−/−fathers show reduced viability in uteroDefects inpi6mutant sperm reflect changes in the abundance of specific mRNAs.

scholarly journals Intergenerational metabolic priming by sperm piRNAs

2021 ◽  
Author(s):  
Adelheid Lempradl ◽  
Unn Kugelberg ◽  
Mary Iconomou ◽  
Ian Beddows ◽  
Daniel Nätt ◽  
...  
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Target Gene ◽  
Epigenetic Inheritance ◽  
Mature Sperm ◽  
Transcriptional Reprogramming ◽  
Complementary Sequences ◽  
Specific Manner ◽  
Dietary Sugar ◽  
Pirna Pathway

Preconception parental environment can reproducibly program offspring phenotype without altering the DNA sequence, yet the mechanisms underpinning this epigenetic inheritance remains elusive. Here, we demonstrate the existence of an intact piRNA-pathway in mature Drosophila sperm and show that pathway modulation alters offspring gene transcription in a sequence-specific manner. We map a dynamic small RNA content in developing sperm and find that the mature sperm carry a highly distinct small RNA cargo. By biochemical pulldown, we identify a small RNA subset bound directly to piwi protein. And, we show that piRNA-pathway controlled sperm small RNAs are linked to target gene repression in offspring. Critically, we find that full piRNA-pathway dosage is necessary for the intergenerational metabolic and transcriptional reprogramming events triggered by high paternal dietary sugar. These data provide a direct link between regulation of endogenous mature sperm small RNAs and transcriptional programming of complementary sequences in offspring. Thus, we identify a novel mediator of paternal intergenerational epigenetic inheritance.

scholarly journals The architecture of SARS-CoV-2 transcriptome

2020 ◽  
Author(s):  
Dongwan Kim ◽  
Joo-Yeon Lee ◽  
Jeong-Sun Yang ◽  
Jun Won Kim ◽  
V. Narry Kim ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Sequencing ◽  
Rna Modification ◽  
List Type ◽  
Rna Modifications ◽  
Subgenomic Rnas ◽  
Frequent Motif ◽  
High Resolution Map ◽  
Functional Investigation ◽  
Resolution Map

AbstractSARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3′ tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.HighlightsWe provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing.The transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical.In addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs.We discover at least 41 potential RNA modification sites with an AAGAA motif.

scholarly journals Multiple roles of PIWIL1 in mouse neocorticogenesis

2017 ◽  
Author(s):  
Barbara Viljetic ◽  
Liyang Diao ◽  
Jixia Liu ◽  
Zeljka Krsnik ◽  
Sagara H.R. Wijeratne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Small Rna ◽  
Radial Glia ◽  
Multiple Roles ◽  
Small Rna Sequencing ◽  
Knock Out ◽  
Deep Layers ◽  
Pirna Pathway ◽  
The Brain

AbstractPIWI-interacting RNAs (piRNAs) and their associated PIWI proteins play an important role in repressing transposable elements in animal germlines. However, little is known about the function of PIWI proteins and piRNAs in the developing brain. Here, we investigated the role of an important PIWI family member, Piwi-like protein 1 (Piwil1; also known as Miwi in mouse) in the developing mouse neocortex. Using a Piwil1 knock-out (Piwil1 KO) mouse strain, we found that Piwil1 is essential for several steps of neocorticogenesis, including neocortical cell cycle, neuron migration and dendritogenesis. Piwil1 deletion resulted in increased cell cycle re-entry at embryonic day 17 (E17) when predominantly intracortically projecting neurons are being produced. Prenatal Piwil1 deletion increased the number of Pax6+ radial glia at postnatal day 0 (P0). Furthermore, Piwil1 deletion disrupted migration of Satb2+ neurons within deep layers at E17, P0 and P7. Satb2+ neurons showed increased co-localization with Bcl11b (also known as Ctip2), marker of subcortically projecting neurons. Piwil1 knockouts had disrupted neocortical circuitry represented by thinning of the corpus callosum and altered dendritogenesis. We further investigated if Piwil1 deletion disrupted expression levels of neocortical piRNAs by small RNA-sequencing in neocortex. We did not find differential expression of piRNAs in the neocortices of Piwil1 KO, while differences were observed in other Piwil1 KO tissues. This result suggests that Piwil1 may act independently of piRNAs and have novel roles in higher cognitive centers, such as neocortex. In addition, we report a screen of piRNAs derived from tRNA fragments in developing neocortices. Our result is the first report of selective subsets of piRNAs and tRNA fragments in developing prenatal neocortices and helps clarify some outstanding questions about the role of the piRNA pathway in the brain.

Small RNA and transcriptome sequencing of a symptomatic peony plant reveals mixed infections with novel viruses

Plant Disease ◽  
2021 ◽  
Author(s):  
Anning Jia ◽  
Chenge Yan ◽  
Hang Yin ◽  
Rui Sun ◽  
Fei Xia ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Triple Gene Block ◽  
Field Investigation ◽  
Mixed Infections ◽  
Species Classification ◽  
Gene Block ◽  
Sequence Identity

To identify the viruses in tree peony plants associated with the symptoms of yellowing, leaf rolling, stunted growth, and decline, high-throughput sequencing of small RNA and mRNA was conducted from a single symptomatic plant. Bioinformatic analyses and reconstruction of viral genomes indicated mixed viral infections involving cycas necrotic stunt virus (CNSV), apple stem grooving virus (ASGV), lychnis mottle virus (LycMoV), grapevine line pattern virus (GLPV), and three new viruses designated as peony yellowing-associated citrivirus (PYaCV, Citrivirus in Betaflexiviridae), peony betaflexivirus 1 (PeV1, unclassified in Betaflexiviridae), and peony leafroll-associated virus (PLRaV, Ampelovirus in Closteroviridae). PYaCV was 8,666 nucleaotides (nt) in length, comprising three open reading frames (ORFs) and shared 63.8–75.9% nucleotide sequence identity with citrus leaf blotch virus (CLBV) isolates. However, the ORF encoding the replication-associated protein (REP) shared 57% and 52% sequence identities at the nt and amino acid (aa) level, respectively, with other reported CLBV isolates, which were below the criterion for species classification within the family Betaflexiviridae. Recombination analysis identified putative recombination sites in PYaCV, which originated from CLBV. PeV1, only identified from the transcriptome data, was 8,124 nt in length with five ORFs encoding the REP (ORF1), triple gene block (TGB, ORF2–4) and coat protein (CP, ORF5) proteins. Phylogenetic analysis and sequence comparison showed that PeV1 clustered with an unassigned member, the garlic yellow mosaic-associated virus (GYMaV) within the Betaflexiviridae family, into a separate clade. Partial genome sequence analysis of PLRaV (12,545 nt) showed it contained seven ORFs encoding the partial polyprotein 1a, the RNA-dependent RNA polymerase (RdRp), two small hydrophobic proteins p11 and p6, HSP70h, p55, and a CP duplicate, which shared low aa sequence identity with Closteroviridae family members. Phylogenetic analysis based on the aa sequences of RdRp or HSP70h indicated that PLRaV clustered with grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-13 in the Ampelovirus genus. Field investigation confirmed the wide distribution of these viruses, causing mixed infections of peony plants in Beijing.

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