scholarly journals Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs

2021 ◽  
Author(s):  
Yu H. Sun ◽  
Ruoqiao Huiyi Wang ◽  
Khai Du ◽  
Jihong Zheng ◽  
Li Huitong Xie ◽  
...  
Keyword(s):  
Small Rnas ◽  
Protein Production ◽  
Open Reading Frames ◽  
Fine Tuning ◽  
Untranslated Regions ◽  
Protein Coding ◽  
Evolutionary Force ◽  
Non Coding Rnas ◽  
Pirna Pathway ◽  
Reading Frames

PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. Previously, we showed that ribosomes guide the biogenesis of piRNAs from long non-coding RNAs (lncRNAs) after translating the short open reading frames (ORFs) near their 5' cap. It remained unclear, however, how ribosomes proceed downstream of ORFs and how piRNA precursors distinguish from other RNAs. It is thus important to test whether a short ORF length is required for substrate recognition for ribosome guided-piRNA biogenesis. Here, we characterized a poorly understood class of piRNAs that originate from the 3' untranslated regions (3'UTRs) of protein coding genes in mice and chickens. We demonstrate that their precursors are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3'UTRs after translation of upstream long ORFs. Similar to non-sense mediated decay (NMD), piRNA biogenesis degrades mRNA right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Interestingly, however, we found that NMD, along with other surveillance pathways for ribosome recycling are temporally sequestered during the pachytene stage to allow for robust piRNA production. Although 3'UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded transposable elements (TEs) and produce piRNAs that cleave TEs, suggesting that TE suppression, rather than the function of proteins, is the primary evolutionary force maintaining a subset of mRNAs as piRNA precursors. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved, general piRNA biogenesis mechanism that recognizes translating RNAs regardless of their ORF length in amniotes.

scholarly journals Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu H. Sun ◽  
Ruoqiao Huiyi Wang ◽  
Khai Du ◽  
Jiang Zhu ◽  
Jihong Zheng ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Transposable Element ◽  
Small Rnas ◽  
Protein Production ◽  
Fine Tuning ◽  
Untranslated Regions ◽  
Protein Coding ◽  
Rna Surveillance ◽  
Non Coding Rnas ◽  
Pirna Pathway

AbstractPIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. piRNAs originate from transposable element (TE) RNAs, long non-coding RNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, with the last being the least characterized of the three piRNA classes. Here, we demonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3´UTRs in mice and chickens. At the pachytene stage, when other co-translational RNA surveillance pathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved piRNA biogenesis mechanism that recognizes translating RNAs in amniotes.

scholarly journals A community-driven roadmap to advance research on translated open reading frames detected by Ribo-seq

2021 ◽  
Author(s):  
Jonathan M Mudge ◽  
Jorge Ruiz-Orera ◽  
John R Prensner ◽  
Marie A Brunet ◽  
Jose Manuel Gonzalez ◽  
...  
Keyword(s):  
Gene Annotation ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Biological Databases ◽  
Protein Coding ◽  
Circular Problem ◽  
Advance Research ◽  
Non Coding Rnas ◽  
Reading Frames

Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational vocabulary of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

scholarly journals RNA G-quadruplexes mark repressive upstream open reading frames in human mRNAs

2017 ◽  
Author(s):  
Pierre Murat ◽  
Giovanni Marsico ◽  
Barbara Herdy ◽  
Avazeh Ghanbarian ◽  
Guillem Portella ◽  
...  
Keyword(s):  
Secondary Structures ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Translation Regulation ◽  
Physical Interaction ◽  
Protein Coding ◽  
Upstream Open Reading Frames ◽  
Nucleotide Resolution ◽  
Reading Frames

ABSTRACTRNA secondary structures in the 5’ untranslated regions (UTRs) of mRNAs have been characterised as key determinants of translation initiation. However the role of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), in modulating translation of human mRNAs and the associated mechanisms remain largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape of human mRNAs with structured 5’ untranslated regions (5’-UTR). We found that inefficiently translated mRNAs, containing rG4-forming sequences in their 5’-UTRs, have an accumulation of ribosome footprints in their 5’-UTRs. We show that rG4-forming sequences are determinants of 5’-UTR translation, suggesting that the folding of rG4 structures thwarts the translation of protein coding sequences (CDS) by stimulating the translation of repressive upstream open reading frames (uORFs). To support our model, we demonstrate that depletion of two rG4s-specialised DEAH-box helicases, DHX36 and DHX9, shifts translation towards rG4-containing uORFs reducing the translation of selected transcripts comprising proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) demonstrate that translation regulation is mediated through direct physical interaction between the helicase and its rG4 substrate. Our findings unveil a previously unknown role for non-canonical structures in governing 5’-UTR translation and suggest that the interaction of helicases with rG4s could be considered as a target for future therapeutic intervention.

scholarly journals Annotating high-impact 5’untranslated region variants with the UTRannotator

2020 ◽  
Author(s):  
Xiaolei Zhang ◽  
Matthew Wakeling ◽  
James Ware ◽  
Nicola Whiffin
Keyword(s):  
Open Reading Frames ◽  
Supplementary Information ◽  
Untranslated Regions ◽  
Protein Coding ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
Upstream Open Reading Frames ◽  
The Impact ◽  
Reading Frames

AbstractSummaryCurrent tools to annotate the predicted effect of genetic variants are heavily biased towards protein-coding sequence. Variants outside of these regions may have a large impact on protein expression and/or structure and can lead to disease, but this effect can be challenging to predict. Consequently, these variants are poorly annotated using standard tools. We have developed a plugin to the Ensembl Variant Effect Predictor, the UTRannotator, that annotates variants in 5’untranslated regions (5’UTR) that create or disrupt upstream open reading frames (uORFs). We investigate the utility of this tool using the ClinVar database, providing an annotation for 30.8% of all 5’UTR (likely) pathogenic variants, and highlighting 31 variants of uncertain significance as candidates for further follow-up. We will continue to update the UTR annotator as we gain new knowledge on the impact of variants in UTRs.Availability and implementationUTRannotator is freely available on Github: https://github.com/ImperialCardioGenetics/UTRannotatorSupplementary informationSupplementary data are available at bioRxiv.

scholarly journals Comprehensive Annotations of Human Herpesvirus 6A and 6B Genomes Reveal Novel and Conserved Genomic Features

2019 ◽  
Author(s):  
Yaara Finkel ◽  
Dominik Schmiedel ◽  
Julie Tai-Schmiedel ◽  
Aharon Nachshon ◽  
Michal Schwartz ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Temporal Expression ◽  
Protein Coding ◽  
Functional Studies ◽  
Viral Genes ◽  
Non Coding Rnas ◽  
Coding Potential ◽  
Reading Frames

AbstractHuman herpesvirus 6 (HHV-6) A and B are highly ubiquitous betaherpesviruses, infecting the majority of the human population. Like other herpesviruses, they encompass large genomes and our understanding of their protein coding potential is far from complete. Here we employ ribosome profiling and systematic transcript analysis to experimentally define the HHV-6 translation products and to follow their temporal expression. We identify hundreds of new open reading frames (ORFs), including many upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. Furthermore, by integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs that are conserved across betaherpesviruses and we show that uORFs are specifically enriched in late viral genes. Using our transcriptome measurements, we identified three highly abundant HHV-6 encoded long non-coding RNAs (lncRNAs), one of which generates a non-polyadenylated stable intron that appears to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features that are conserved between betaherpesviruses, providing a rich resource for future functional studies.

scholarly journals Annotating high-impact 5′untranslated region variants with the UTRannotator

2020 ◽  
Author(s):  
Xiaolei Zhang ◽  
Matthew Wakeling ◽  
James Ware ◽  
Nicola Whiffin
Keyword(s):  
Open Reading Frames ◽  
Supplementary Information ◽  
Untranslated Regions ◽  
Protein Coding ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
Upstream Open Reading Frames ◽  
The Impact ◽  
Reading Frames

Abstract Summary Current tools to annotate the predicted effect of genetic variants are heavily biased towards protein-coding sequence. Variants outside of these regions may have a large impact on protein expression and/or structure and can lead to disease, but this effect can be challenging to predict. Consequently, these variants are poorly annotated using standard tools. We have developed a plugin to the Ensembl Variant Effect Predictor, the UTRannotator, that annotates variants in 5′untranslated regions (5′UTR) that create or disrupt upstream open reading frames. We investigate the utility of this tool using the ClinVar database, providing an annotation for 31.9% of all 5′UTR (likely) pathogenic variants, and highlighting 31 variants of uncertain significance as candidates for further follow-up. We will continue to update the UTRannotator as we gain new knowledge on the impact of variants in UTRs. Availability and implementation UTRannotator is freely available on Github: https://github.com/ImperialCardioGenetics/UTRannotator. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Visualizing the periodic Ribo-seq reads with RiboPlotR

2019 ◽  
Author(s):  
Hsin-Yen Larry Wu ◽  
Polly Yingshan Hsu
Keyword(s):  
Mrna Translation ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Rna Seq ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Non Coding Rnas ◽  
Reading Frames ◽  
Small Open Reading Frames

ABSTRACTBackgroundRibo-seq has revolutionized the study of mRNA translation in a genome-wide scale. High-quality Ribo-seq data display strong 3-nucleotide (nt) periodicity, which corresponds to translating ribosomes decipher three nucleotides each time. While the 3-nt periodicity has been widely used to study novel translation events and identify small open reading frames on presumed non-coding RNAs, tools which allow the visualization of those events remain underdeveloped.FindingsRiboPlotR is a visualization package written in R that presents both RNA-seq coverage and Ribo-seq reads for all annotated transcript isoforms in a context of a given gene. In particular, RiboPlotR plots Ribo-seq reads mapped in three reading frames using three colors for one isoform model at a time. Moreover, RiboPlotR shows Ribo-seq reads on upstream ORFs, 5’ and 3’ untranslated regions and introns, which is critical for observing new translation events and potential regulatory mechanisms.ConclusionsRiboPlotR is freely available (https://github.com/hsinyenwu/RiboPlotR) and allows the visualization of the translating features in Ribo-seq data.

scholarly journals Comprehensive annotations of human herpesvirus 6A and 6B genomes reveal novel and conserved genomic features

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Yaara Finkel ◽  
Dominik Schmiedel ◽  
Julie Tai-Schmiedel ◽  
Aharon Nachshon ◽  
Roni Winkler ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Protein Coding ◽  
Genomic Features ◽  
Functional Studies ◽  
Viral Genes ◽  
Non Coding Rnas ◽  
Coding Potential ◽  
Reading Frames

Human herpesvirus-6 (HHV-6) A and B are ubiquitous betaherpesviruses, infecting the majority of the human population. They encompass large genomes and our understanding of their protein coding potential is far from complete. Here, we employ ribosome-profiling and systematic transcript-analysis to experimentally define HHV-6 translation products. We identify hundreds of new open reading frames (ORFs), including upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. By integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs conserved across betaherpesviruses and we show uORFs are enriched in late viral genes. We identified three highly abundant HHV-6 encoded long non-coding RNAs, one of which generates a non-polyadenylated stable intron appearing to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features conserved between betaherpesviruses, providing a rich resource for future functional studies.

scholarly journals Long non-coding RNAs as a source of new peptides

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jorge Ruiz-Orera ◽  
Xavier Messeguer ◽  
Juan Antonio Subirana ◽  
M Mar Alba
Keyword(s):  
Transcriptome Sequencing ◽  
De Novo ◽  
Large Fraction ◽  
Open Reading Frames ◽  
Protein Coding ◽  
Coding Sequences ◽  
Non Coding Rnas ◽  
Sequence Constraints ◽  
Coding Potential ◽  
Reading Frames

Deep transcriptome sequencing has revealed the existence of many transcripts that lack long or conserved open reading frames (ORFs) and which have been termed long non-coding RNAs (lncRNAs). The vast majority of lncRNAs are lineage-specific and do not yet have a known function. In this study, we test the hypothesis that they may act as a repository for the synthesis of new peptides. We find that a large fraction of the lncRNAs expressed in cells from six different species is associated with ribosomes. The patterns of ribosome protection are consistent with the translation of short peptides. lncRNAs show similar coding potential and sequence constraints than evolutionary young protein coding sequences, indicating that they play an important role in de novo protein evolution.

scholarly journals Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Robin-Lee Troskie ◽  
Yohaann Jafrani ◽  
Tim R. Mercer ◽  
Adam D. Ewing ◽  
Geoffrey J. Faulkner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Open Reading Frames ◽  
Cdna Sequencing ◽  
Protein Coding ◽  
Dynamic Component ◽  
Gene Copies ◽  
Long Read ◽  
Normal Human ◽  
Reading Frames ◽  
Transcriptional Landscape

AbstractPseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape.

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