Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA-counterpart in terminal hematopoiesis

Each day, about 1012 erythrocytes and platelets are released into the blood stream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs amongst hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA-counterpart. We found that only 1 out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of 1000s of identified putative open reading frames, deep ribosome-footprinting sequencing and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.

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Abundant circular RNA expression in terminal myeloid differentiation does not correlate with frequent circRNA translation, or with circRNA-mediated translation regulation

10.1101/2020.10.22.348276 ◽

2020 ◽

Author(s):

Benoit P. Nicolet ◽

Sjoert B.G. Jansen ◽

Esther Heideveld ◽

Willem H. Ouwehand ◽

Emile van den Akker ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Hematopoietic Cells ◽

Blood Stream ◽

Mass Spectrometry Analysis ◽

Translation Regulation ◽

Circular Rnas ◽

Hematopoietic Stem ◽

Erythroid Precursor ◽

Spectrometry Analysis

ABSTRACTEach day, about 1012 erythrocytes and platelets are released into the blood stream. This substantial output from hematopoietic stem cells is tightly regulated by transcription factors and epigenetic modifications. Whether and how non-coding RNAs such as circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not well understood. We recently reported a circRNA expression map of hematopoietic cells, revealing that erythrocytes and platelets contain the highest levels and numbers of circRNAs. Here, we provide the first detailed and comprehensive analysis of circRNA expression during red blood cell and megakaryocyte differentiation. CircRNA expression significantly increased during erythroid precursor differentiation into red blood cells and in differentiating megakaryocytes, in particular upon enucleation. To determine if circRNAs can modulate hematopoietic differentiation, we compared the expression levels of circRNAs and mRNAs with that of ribosomal foot printing read. This multi-omics approach revealed that only 20 out of 748 (2.6%) circRNAs associated with translation regulation of their mRNA counterparts. Furthermore, irrespective of the thousands of identified putative open reading frames in circRNAs, deep ribosome-footprinting sequencing and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs in erythroblasts, megakaryocytes and platelets. In conclusion, circRNAs in platelets and red blood cells are highly abundant and alter their expression profile during differentiation, yet their contribution to regulate cellular processes remains enigmatic.

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Molecular Characterization and Genomic Analysis of Novel Phage vB_ ShiP-A7 Infecting Multidrug-Resistant Shiglla Fexneri and Escherichia Coli

10.21203/rs.3.rs-96088/v1 ◽

2020 ◽

Author(s):

Jing Xu ◽

Yu Gu ◽

Xinyan Yu ◽

Ruiyang Zhang ◽

Xuesen Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Case Reports ◽

Burst Size ◽

Shigella Flexneri ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Open Reading Frames ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Spherical Head

Abstract BackgroundPhage therapy has regained more attention due to the rise of multidrug-resistant (MDR) bacteria. Several case reports demonstrated clinical application of phage in resolving infections caused by MDR bacteria in recent years. ResultsWe isolated a new phage, vB_ShiP-A7, and then investigated its characteristics. Phage vB_ShiP-A7 is a member of Podoviridae that has an icosahedral spherical head and a short tail. vB_ShiP-A7 has large burst size and short replication time. vB_ShiP-A7’s genome is linear double stranded DNA composed of 40058 bp, encoding forty-three putative open reading frames. Comparative genome analysis demonstrated vB_ShiP-A7’s genome sequence is closely related to fifteen different phages (coverage 74-88%, identity 86-93%). Mass Spectrometry analysis revealed that twelve known proteins and six hypothetical proteins exist in particles of vB_ShiP-A7. Genome and proteome analyses confirmed the absence of lysogen-related proteins and toxic proteins in this phage. In addition, phage vB_ShiP-A7 can significantly reduce the growth of clinical MDR stains of Shigella ﬂexneri and Escherichia coli in liquid culture. Furthermore, vB_ShiP-A7 can disrupt biofilms formed by Shigella ﬂexneri or Escherichia coli in vitro. ConclusionPhage vB_ShiP-A7 is a stable novel phage, which has a strong application potential to inhibit MDR stains of Shigella ﬂexneri and Escherichia coli. Comparing the genomes between vB_ShiP-A7 and other closely-related phages will help us better understand the evolutionary mechanism of phages.

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RiboToolkit: an integrated platform for analysis and annotation of ribosome profiling data to decode mRNA translation at codon resolution

Nucleic Acids Research ◽

10.1093/nar/gkaa395 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W218-W229 ◽

Cited By ~ 1

Author(s):

Qi Liu ◽

Tanya Shvarts ◽

Piotr Sliz ◽

Richard I Gregory

Keyword(s):

Mrna Translation ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Translational Efficiency ◽

Translation Efficiency ◽

Sequencing Data ◽

Experimental Conditions ◽

Web Based ◽

Rna Translation ◽

Web Interfaces

Abstract Ribosome profiling (Ribo-seq) is a powerful technology for globally monitoring RNA translation; ranging from codon occupancy profiling, identification of actively translated open reading frames (ORFs), to the quantification of translational efficiency under various physiological or experimental conditions. However, analyzing and decoding translation information from Ribo-seq data is not trivial. Although there are many existing tools to analyze Ribo-seq data, most of these tools are designed for specific or limited functionalities and an easy-to-use integrated tool to analyze Ribo-seq data is lacking. Fortunately, the small size (26–34 nt) of ribosome protected fragments (RPFs) in Ribo-seq and the relatively small amount of sequencing data greatly facilitates the development of such a web platform, which is easy to manipulate for users with or without bioinformatic expertise. Thus, we developed RiboToolkit (http://rnabioinfor.tch.harvard.edu/RiboToolkit), a convenient, freely available, web-based service to centralize Ribo-seq data analyses, including data cleaning and quality evaluation, expression analysis based on RPFs, codon occupancy, translation efficiency analysis, differential translation analysis, functional annotation, translation metagene analysis, and identification of actively translated ORFs. Besides, easy-to-use web interfaces were developed to facilitate data analysis and intuitively visualize results. Thus, RiboToolkit will greatly facilitate the study of mRNA translation based on ribosome profiling.

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Determinants of genome-wide distribution and evolution of uORFs in eukaryotes

Nature Communications ◽

10.1038/s41467-021-21394-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hong Zhang ◽

Yirong Wang ◽

Xinkai Wu ◽

Xiaolu Tang ◽

Changcheng Wu ◽

...

Keyword(s):

Driving Forces ◽

Mrna Translation ◽

Purifying Selection ◽

Wide Distribution ◽

Open Reading Frames ◽

Translation Regulation ◽

Effective Population ◽

Upstream Open Reading Frames ◽

Functional Peptides ◽

Regulatory Functions

AbstractUpstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze ~17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (Ne), has shaped the contents of uORFs in eukaryotes. Besides, gene expression level is important in influencing uORF occurrences across genes in a species. Our analyses suggest that most uORFs might play regulatory roles rather than encode functional peptides. We also show that the Kozak sequence context of uORFs has evolved across eukaryotic clades, and that noncanonical uORFs tend to have weaker suppressive effects than canonical uORFs in translation regulation. This study provides insights into the driving forces underlying uORF evolution in eukaryotes.

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Arginine Methylation Of RBM15 By PRMT1 Controls Alternative Splicing Of Genes Involved In Megakaryocyte Differentiation

Blood ◽

10.1182/blood.v122.21.2443.2443 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2443-2443

Author(s):

Xinyang Zhao ◽

Li Zhang ◽

Rui Wang ◽

Ngoc Tung Trans ◽

Hairui Su ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Conflicts Of Interest ◽

Chromosome Translocation ◽

Arginine Methylation ◽

Mass Spectrometry Analysis ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Spectrometry Analysis ◽

Megakaryocyte Differentiation

Abstract More than 90% of under one year old infants with acute megakaryoblastic leukemia (AMKL) have chromosome translocation t(1;22)(p13;q13) with RBM15 fused to MKL1. RBM15 encodes an RNA binding protein important for hematopoietic stem cell self-renewal and differentiation. In agreement with its roles in AMKL, RBM15 is required for normal megakaryocyte differentiation. We found that higher expression of PRMT1 (Protein Arginine Methyltransferase) is commonly seen in M7 leukemia patient samples than other types of myeloid leukemia and that RBM15 is a bona fide methylation target for PRMT1. Using mass spectrometry analysis, we mapped the PRMT1 mediated mono-methylated site. The enzymatic activity of the PRMT1 V2 isoform is required for RBM15 degradation, as both shRNA molecules knocking down PRMT1 and small chemical PRMT1 inhibitors stabilize the RBM15 protein. Mutation of the methylation site to lysine blocks the ubiquitylation mediated degradation. Thus the degradation is a methylation dependent process. We identified the E3 ligase responsible for the degradation. Down-regulation of the RBM15 protein changes the isoform ratio of genes including GATA1 critical megakaryocyte differentiation. We found that RBM15 regulates its interaction with SF3B1A in methylation dependent manner during alternative splicing of GATA1 pre-mRNA. Thus, via methylation triggered RBM15 degradation, the megakaryocyte progenitor cells maintain a delicate balance between differentiation and proliferation by keeping the proper ratio of GATA1s and GATA1-full length mRNA. SF3B1A has been shown to be mutated in myeloid dysplasia syndrome and in several different types of leukemia. Methylation by PRMT1 links the two types of leukemic genes into a single pathway. Our results imply that targeting PRMT1/RBM15 pathway might be a potential therapy for AMKL and other blood malignancies. Disclosures: No relevant conflicts of interest to declare.

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circPDE4B prevents articular cartilage degeneration and promotes repair by acting as a scaffold for RIC8A and MID1

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-219969 ◽

2021 ◽

pp. annrheumdis-2021-219969

Author(s):

Shuying Shen ◽

Yute Yang ◽

Panyang Shen ◽

Jun Ma ◽

Bin Fang ◽

...

Keyword(s):

Rna Binding ◽

Cartilage Degeneration ◽

Mitogen Activated Protein Kinase ◽

Mass Spectrometry Analysis ◽

Circular Rnas ◽

Nucleotide Exchange ◽

Spectrometry Analysis

ObjectivesCircular RNAs (circRNAs) have emerged as significant biological regulators. Herein, we aimed to elucidate the role of an unidentified circRNA (circPDE4B) that is reportedly downregulated in osteoarthritis (OA) tissues.MethodsThe effects of circPDE4B were explored in human and mouse chondrocytes in vitro. Specifically, RNA pull-down (RPD)-mass spectrometry analysis (MS), immunoprecipitation, glutathione-S-transferase (GST) pull-down, RNA immunoprecipitation and RPD assays were performed to verify the interactions between circPDE4B and the RIC8 guanine nucleotide exchange factor A (RIC8A)/midline 1 (MID1) complex. A mouse model of OA was also employed to confirm the role of circPDE4B in OA pathogenesis in vivo.ResultscircPDE4B regulates chondrocyte cell viability and extracellular matrix metabolism. Mechanistically, FUS RNA binding protein (FUS) was found to promote the splicing of circPDE4B, while downregulation of circPDE4B in OA is partially caused by upstream inhibition of FUS. Moreover, circPDE4B facilitates the association between RIC8A and MID1 by acting as a scaffold to promote RIC8A degradation through proteasomal degradation. Furthermore, ubiquitination of RIC8A at K415 abrogates RIC8A degradation. The circPDE4B–RIC8A axis was observed to play an important role in regulating downstream p38 mitogen-activated protein kinase (MAPK) signalling. Furthermore, delivery of a circPDE4B adeno-associated virus (AAV) abrogates the breakdown of cartilage matrix by medial meniscus destabilisation in mice, whereas a RIC8A AAV induces the opposite effect.ConclusionThis work highlights the function of the circPDE4B–RIC8A axis in OA joints, as well as its regulation of MAPK-p38, suggesting this axis as a potential therapeutic target for OA.

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Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

10.1101/806364 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jonathan Bohlen ◽

Kai Fenzl ◽

Günter Kramer ◽

Bernd Bukau ◽

Aurelio A. Teleman

Keyword(s):

Translation Initiation ◽

Human Cells ◽

Open Reading Frames ◽

Translation Regulation ◽

List Type ◽

Translation Efficiency ◽

Initiation Factors ◽

Upstream Open Reading Frames

SUMMARYTranslation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ∼12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.HIGHLIGHTSSelective 40S FPing visualizes regulation of translation initiation on mRNAs in vivoScanning ribosomes are cap-tethered in human cellsOnly one ribosome scans a 5’UTR at a time in human cellsRibosomes retain eIFs during early translation, allowing reinitiation after uORFs

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Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses

Circulation Research ◽

10.1161/circresaha.121.320029 ◽

2021 ◽

Author(s):

Andreas W Heumüller ◽

Alisha Nicole Jones ◽

André Mourão ◽

Marius Klangwart ◽

Chenyue Shi ◽

...

Keyword(s):

Endothelial Cells ◽

Laminar Flow ◽

Affinity Purification ◽

Mass Spectrometry Analysis ◽

Circular Rnas ◽

Cell Orientation ◽

Spectrometry Analysis ◽

Functional Conservation

Background : Circular RNAs (circRNAs) are generated by back-splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. Methods: Mechanistically, we identified the protein syndesmos (SDOS) to specifically interact with cZNF292 in endothelial cells by RNA affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganisation thereby recapitulating cZfp292 phenotypes. Results: Combining published endothelial RNA sequencing datasets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localisation and focal adhesion organisation. Conclusion: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

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Mass spectrometry analysis of mouse hematopoietic stem cells and their progenitors reveals differential expression within and between proteome and transcriptome throughout adult and aged hematopoiesis

10.1101/836692 ◽

2019 ◽

Author(s):

Balyn W. Zaro ◽

Joseph J. Noh ◽

Victoria L. Mascetti ◽

Janos Demeter ◽

Benson M. George ◽

...

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Protein Expression ◽

Hematopoietic Stem Cells ◽

Differential Expression ◽

Cell Types ◽

Mass Spectrometry Analysis ◽

Hematopoietic Stem ◽

Spectrometry Analysis ◽

Mass Spectrometry Technology

SummaryHematopoietic stem cells (HSCs) are responsible for the generation of blood and immune cells throughout life. They have the unique ability to self-renew and generate more HSCs or differentiate into a progenitor cell in response to cell-intrinsic and -extrinsic stimuli. The balance of HSC fate commitment is critical for a healthy blood supply. Imbalances during hematopoiesis, which are frequent in aging, can result in hematological malignancies and pre-malignancies as well as increase risk of atherosclerosis. Given the importance of HSCs and their progenitors, they have been extensively characterized in genomic and transcriptomic studies. However, an understanding of protein expression within the HSC compartment and more broadly throughout hematopoiesis remains poorly understood, and it has been widely reported that the correlation between mRNA and proteins is more complicated than previously thought. Previous mouse mass spectrometry studies have focused either specifically on stem and the first early progenitor or broadly across mixed populations of stem and progenitor cells, which do not allow for cell-type specific protein resolution across stages of differentiation. Mass cytometry has been employed to characterize transcription factor expression in human HSCs and progenitors but does not apply an unbiased discovery approach. New mass spectrometry technology now allows for deep proteomic coverage with no more than 200 ng of sample input. We report here a proteomics resource characterizing protein expression in mouse adult and aged HSCs, multipotent progenitors and oligopotent progenitors, 12 cell types in total. We validated differential expression by flow cytometry analysis and immunofluorescence staining. Additionally, we investigated the relationship between mRNA and protein levels of individual genes in HSCs compared to progenitors through RNA sequencing studies and identified two proteins that appear to be uniquely regulated in the HSC compartment, Cpin1 and Adnp. In summary, this resource provides proteomic coverage of adult and aged hematopoietic stem cells and their progenitors and reveals changes in protein abundance between cell types, with potential future implications in understanding mechanisms for stem-cell maintenance, niche interactions and fate determination.

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LeishIF4E-5 Is a Promastigote-Specific Cap-Binding Protein in Leishmania

International Journal of Molecular Sciences ◽

10.3390/ijms22083979 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3979

Author(s):

Rohit Shrivastava ◽

Nitin Tupperwar ◽

Bar Schwartz ◽

Nofar Baron ◽

Michal Shapira

Keyword(s):

Environmental Conditions ◽

Binding Protein ◽

Binding Pocket ◽

Binding Activity ◽

Mass Spectrometry Analysis ◽

Sand Fly ◽

Translation Regulation ◽

Binding Motif ◽

Spectrometry Analysis ◽

Cytochemical Analysis

Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors’ alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

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