scholarly journals Functional insights from a surface antigen mRNA-bound proteome

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Larissa Melo do Nascimento ◽  
Franziska Egler ◽  
Katharina Arnold ◽  
Nina Papavasiliou ◽  
Christine Clayton ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Rna Binding ◽  
Antigenic Variation ◽  
Affinity Purification ◽  
Surface Glycoprotein ◽  
Cell Protein ◽  
Cis Acting ◽  
Messenger Ribonucleoprotein ◽  
Biological Insight ◽  
Associated Proteins

Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites' Variant Surface Glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2, an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stability, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonucleoprotein particle and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.

scholarly journals The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

2020 ◽  
Author(s):  
Larissa Melo do Nascimento ◽  
Franziska Egler ◽  
Katharina Arnold ◽  
Nina Papavisiliou ◽  
Christine Clayton ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Rna Binding ◽  
Antigenic Variation ◽  
Binding Protein ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Binding Domains ◽  
Associated Proteins ◽  
Polymerase I ◽  
Mrna Binding

Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases. The “bloodstream forms”, which replicate extracellularly in the blood and tissue fluids of mammals, are coated by a monolayer of Variant Surface Glycoprotein (VSG). Switching of the expressed VSG gene is central to parasite pathogenicity because it enables the parasites to evade adaptive immunity via antigenic variation. Adequate levels of VSG expression - 10% of total protein and 7% of mRNA - are attained through very active RNA polymerase I transcription, efficient mRNA processing (trans splicing of a capped leader and polyadenylation), and high mRNA stability. We here show how VSG mRNA stability is maintained. Purification of the VSG mRNA with associated proteins specifically selected CFB2, an F-box mRNA-binding protein that lacks known RNA-binding domains. CFB2 binds to a stabilizing complex (MKT1-PBP1-XAC1-LSM12) that recruits poly(A) binding protein and a specialized cap-binding translation initiation complex, EIF4E6-EIF4G5. The interaction of CFB2 with MKT1 is essential for CFB2’s expression-promoting activity, while the F-box auto-regulates CFB2 abundance via interaction with SKP1, a component of the ubiquitination machinery. The results of reporter experiments indicate that CFB2 acts via conserved sequences in the VSG mRNA 3’-untranslated region. Depletion of CFB2 leads to highly specific loss of VSG mRNA. VSG expression is essential not only for antigenic variation but also for trypanosome cell division. Correspondingly, depletion of CFB2 causes cell cycle arrest, dramatic morphological abnormalities and trypanosome death.

Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability

2017 ◽  
Vol 474 (10) ◽  
pp. 1669-1687
Author(s):  
Hiromi Motohashi ◽  
Yoshiki Mukudai ◽  
Chihiro Ito ◽  
Kosuke Kato ◽  
Toshikazu Shimane ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
T Cell ◽  
Growth Factor ◽  
Mrna Stability ◽  
Rna Binding ◽  
Rna Degradation ◽  
Related Protein ◽  
Binding Ability ◽  
Cis Acting ◽  
Post Transcriptional Regulation

Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3′-untranslated region (3′-UTR) of TPD52 as a cis-acting element in post-transcriptional gene regulation. Several deletion mutants of the 3′-UTR of TPD52 mRNA were constructed and ligated to the 3′-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis-acting region, located in the 78-280 region of the 5′-proximal region of the 3′-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3′-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis-acting element and trans-acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.

scholarly journals Possible involvement of messenger RNA-associated proteins in protein synthesis.

1982 ◽  
Vol 93 (3) ◽  
pp. 893-898 ◽  
Author(s):  
H P Schmid ◽  
K Köhler ◽  
B Setyono
Keyword(s):  
Messenger Rna ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ascites Cell ◽  
Free System ◽  
Molecular Weights ◽  
Messenger Ribonucleoprotein ◽  
Associated Proteins ◽  
Globin Synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.

scholarly journals RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

2021 ◽  
Vol 7 (1) ◽  
pp. 11 ◽  
Author(s):  
André P. Gerber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Cell Protein ◽  
Transcriptional Regulatory Networks ◽  
Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

scholarly journals RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xia Wang ◽  
Jin Wang ◽  
Yu-Man Tsui ◽  
Chaoran Shi ◽  
Ying Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mrna Stability ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Molecular Characteristics ◽  
Specific Gene ◽  
Functional Studies ◽  
Poor Differentiation ◽  
Development In Vitro

AbstractGrowing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-β2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-β signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

scholarly journals Targeting the Variable Surface of African Trypanosomes with Variant Surface Glycoprotein-Specific, Serum-Stable RNA Aptamers

2003 ◽  
Vol 2 (1) ◽  
pp. 84-94 ◽  
Author(s):  
Mihaela Lorger ◽  
Markus Engstler ◽  
Matthias Homann ◽  
H. Ulrich Göringer
Keyword(s):  
Antigenic Variation ◽  
Sleeping Sickness ◽  
Rna Aptamers ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Parasite Protein ◽  
African Trypanosomes ◽  
Variable Surface ◽  
New Strategy

ABSTRACT African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.

scholarly journals Characterization of a beta-actin mRNA zipcode-binding protein.

1997 ◽  
Vol 17 (4) ◽  
pp. 2158-2165 ◽  
Author(s):  
A F Ross ◽  
Y Oleynikov ◽  
E H Kislauskis ◽  
K L Taneja ◽  
R H Singer
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Nuclear Export Signal ◽  
Degenerate Primers ◽  
Band Shift ◽  
Beta Actin ◽  
Actin Mrna

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

scholarly journals Comprehensive landscape and future perspectives of circular RNAs in colorectal cancer

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fei Long ◽  
Zhi Lin ◽  
Liang Li ◽  
Min Ma ◽  
Zhixing Lu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Circular Rnas ◽  
Future Perspectives ◽  
Cis Acting ◽  
New Class ◽  
Functional Peptides ◽  
Mirna Sponges

AbstractColorectal cancer (CRC) is a common hereditary tumor that is often fatal. Its pathogenesis involves multiple genes, including circular RNAs (circRNAs). Notably, circRNAs constitute a new class of noncoding RNAs (ncRNAs) with a covalently closed loop structure and have been characterized as stable, conserved molecules that are abundantly expressed in tissue/development-specific patterns in eukaryotes. Based on accumulating evidence, circRNAs are aberrantly expressed in CRC tissues, cells, exosomes, and blood from patients with CRC. Moreover, numerous circRNAs have been identified as either oncogenes or tumor suppressors that mediate tumorigenesis, metastasis and chemoradiation resistance in CRC. Although the regulatory mechanisms of circRNA biogenesis and functions remain fairly elusive, interesting results have been obtained in studies investigating CRC. In particular, the expression of circRNAs in CRC is comprehensively modulated by multiple factors, such as splicing factors, transcription factors, specific enzymes and cis-acting elements. More importantly, circRNAs exert pivotal effects on CRC through various mechanisms, including acting as miRNA sponges or decoys, interacting with RNA binding proteins, and even translating functional peptides. Finally, circRNAs may serve as promising diagnostic and prognostic biomarkers and potential therapeutic targets in the clinical practice of CRC. In this review, we discuss the dysregulation, functions and clinical significance of circRNAs in CRC and further discuss the molecular mechanisms by which circRNAs exert their functions and how their expression is regulated. Based on this review, we hope to reveal the functions of circRNAs in the initiation and progression of cancer and highlight the future perspectives on strategies targeting circRNAs in cancer research.

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