Active role of free RNA during the early phase of proteostasis stress

ABSTRACTTransient sequestration of proteins and RNA is an essential principle of cellular reaction to stress. Compared to polypeptides, less is known about the role of RNA released from polysomes during acute proteostasis stress. Using quantitative mass spectrometry, we identified a set of proteins assembled by free RNA in the heat-shocked mammalian cytosol. RNA-associated proteins displayed higher disorder and larger size, which supports the role of multivalent interactions during the initial phase of the RNA granule formation. Structural features of the free RNA interactors defined them as a subset of RNA-binding proteins. The interactome contained preferentially the active form of eIF2α. The interaction between assembled proteins in vivo required RNA. The reconstitution of the association process in vitro indicated to the multimolecular basis for the increased binding to RNA upon heat shock in the cytosol. Our results reveal how free RNA can participate in reorganization of cellular functions during proteostasis stress.

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Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz086 ◽

2019 ◽

Vol 11 (10) ◽

pp. 845-859 ◽

Cited By ~ 10

Author(s):

Alisha N Jones ◽

Michael Sattler

Keyword(s):

High Resolution ◽

Structural Biology ◽

Recent Progress ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Structural Features ◽

Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

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YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

The Journal of Cell Biology ◽

10.1083/jcb.201411047 ◽

2015 ◽

Vol 208 (7) ◽

pp. 913-929 ◽

Cited By ~ 101

Author(s):

Syam Prakash Somasekharan ◽

Amal El-Naggar ◽

Gabriel Leprivier ◽

Hongwei Cheng ◽

Shamil Hajee ◽

...

Keyword(s):

Tumor Progression ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Granule Formation ◽

Ribonucleoprotein Complexes ◽

Highly Correlated ◽

As Stress

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.

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Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01882-1 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zizhen Si ◽

Lei Yu ◽

Haoyu Jing ◽

Lun Wu ◽

Xidi Wang

Keyword(s):

Colorectal Cancer ◽

Rna Binding ◽

Xenograft Model ◽

Luciferase Reporter ◽

Cancer Proliferation ◽

Mouse Xenograft ◽

Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

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Identification of mRNAs Associated with αCP2-Containing RNP Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.7055-7067.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 7055-7067 ◽

Cited By ~ 45

Author(s):

Shelly A. Waggoner ◽

Stephen A. Liebhaber

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Cell Function ◽

Rna Binding Proteins ◽

Translation Control ◽

Viral Mrnas ◽

Posttranscriptional Gene Regulation ◽

Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

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SSMART: Sequence-structure motif identification for RNA-binding proteins

10.1101/287953 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alina Munteanu ◽

Neelanjan Mukherjee ◽

Uwe Ohler

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Motif ◽

Primary Sequence ◽

Sequence Structure ◽

Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

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A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

Molecular and Cellular Biology ◽

10.1128/mcb.7.9.3268 ◽

1987 ◽

Vol 7 (9) ◽

pp. 3268-3276 ◽

Cited By ~ 245

Author(s):

A B Sachs ◽

R W Davis ◽

R D Kornberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Association Constant ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

High Affinity ◽

Terminal Domain ◽

Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

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A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3668 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3668-3678 ◽

Cited By ~ 110

Author(s):

M F Henry ◽

P A Silver

Keyword(s):

Normal Cell ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Temperature Sensitive ◽

Arginine Residues ◽

Cognate Gene ◽

Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes.

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The Emerging Role and Promise of Circular RNAs in Obesity and Related Metabolic Disorders

Cells ◽

10.3390/cells9061473 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1473

Author(s):

Mohamed Zaiou

Keyword(s):

Rna Binding ◽

Metabolic Diseases ◽

Rna Binding Proteins ◽

In Vivo Studies ◽

Future Research ◽

Circular Rnas ◽

Exciting Field ◽

Rna Molecules

Circular RNAs (circRNAs) are genome transcripts that are produced from back-splicing of specific regions of pre-mRNA. These single-stranded RNA molecules are widely expressed across diverse phyla and many of them are stable and evolutionary conserved between species. Growing evidence suggests that many circRNAs function as master regulators of gene expression by influencing both transcription and translation processes. Mechanistically, circRNAs are predicted to act as endogenous microRNA (miRNA) sponges, interact with functional RNA-binding proteins (RBPs), and associate with elements of the transcriptional machinery in the nucleus. Evidence is mounting that dysregulation of circRNAs is closely related to the occurrence of a range of diseases including cancer and metabolic diseases. Indeed, there are several reports implicating circRNAs in cardiovascular diseases (CVD), diabetes, hypertension, and atherosclerosis. However, there is very little research addressing the potential role of these RNA transcripts in the occurrence and development of obesity. Emerging data from in vitro and in vivo studies suggest that circRNAs are novel players in adipogenesis, white adipose browning, obesity, obesity-induced inflammation, and insulin resistance. This study explores the current state of knowledge on circRNAs regulating molecular processes associated with adipogenesis and obesity, highlights some of the challenges encountered while studying circRNAs and suggests some perspectives for future research directions in this exciting field of study.

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Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

Biomolecules ◽

10.3390/biom10060872 ◽

2020 ◽

Vol 10 (6) ◽

pp. 872 ◽

Cited By ~ 1

Author(s):

Clemens Grimm ◽

Jann-Patrick Pelz ◽

Cornelius Schneider ◽

Katrin Schäffler ◽

Utz Fischer

Keyword(s):

Crystal Structure ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Turnover Rates ◽

Mrna Transcription ◽

Terminal Part ◽

Protein Output

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

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POLIII-derived non-coding RNAs acting as scaffolds and decoys

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz049 ◽

2019 ◽

Vol 11 (10) ◽

pp. 880-885 ◽

Cited By ~ 5

Author(s):

Hendrik Täuber ◽

Stefan Hüttelmaier ◽

Marcel Köhn

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Current Knowledge ◽

Cellular Functions ◽

Control Of Gene Expression ◽

Ribonucleoprotein Complexes ◽

Small Ncrnas ◽

Non Coding Rnas ◽

And Function

Abstract A large variety of eukaryotic small structured POLIII-derived non-coding RNAs (ncRNAs) have been described in the past. However, for only few, e.g. 7SL and H1/MRP families, cellular functions are well understood. For the vast majority of these transcripts, cellular functions remain unknown. Recent findings on the role of Y RNAs and other POLIII-derived ncRNAs suggest an evolutionarily conserved function of these ncRNAs in the assembly and function of ribonucleoprotein complexes (RNPs). These RNPs provide cellular `machineries’, which are essential for guiding the fate and function of a variety of RNAs. In this review, we summarize current knowledge on the role of POLIII-derived ncRNAs in the assembly and function of RNPs. We propose that these ncRNAs serve as scaffolding factors that `chaperone’ RNA-binding proteins (RBPs) to form functional RNPs. In addition or associated with this role, some small ncRNAs act as molecular decoys impairing the RBP-guided control of RNA fate by competing with other RNA substrates. This suggests that POLIII-derived ncRNAs serve essential and conserved roles in the assembly of larger RNPs and thus the control of gene expression by indirectly guiding the fate of mRNAs and lncRNAs.

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