scholarly journals Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

2017 ◽  
Vol 114 (9) ◽  
pp. 2206-2211 ◽  
Author(s):  
Niyati Jain ◽  
Hsuan-Chun Lin ◽  
Christopher E. Morgan ◽  
Michael E. Harris ◽  
Blanton S. Tolbert
Keyword(s):  
Rna Binding ◽  
Sequence Motif ◽  
Hnrnp A1 ◽  
Loop Analysis ◽  
Splicing Silencer ◽  
Rna Ligands ◽  
Apical Loop ◽  
Affinity Distribution

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

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

2018 ◽  
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]

scholarly journals The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

1997 ◽  
Vol 17 (6) ◽  
pp. 3194-3201 ◽  
Author(s):  
R J Buckanovich ◽  
R B Darnell
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Motor Dysfunction ◽  
High Affinity ◽  
Loop Region ◽  
Nuclear Rna ◽  
Rna Ligands

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

Coordinate regulation of heterogeneous nuclear ribonucleoprotein dynamics by steroid hormones in the human fallopian tube and endometrium in vivo and in vitro

2012 ◽  
Vol 302 (10) ◽  
pp. E1269-E1282 ◽  
Author(s):  
Ruijin Shao ◽  
Xiaoqin Wang ◽  
Birgitta Weijdegård ◽  
Anders Norström ◽  
Julia Fernandez-Rodriguez ◽  
...  
Keyword(s):  
Steroid Hormones ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intracellular Localization ◽  
Hnrnp A1 ◽  
Fallopian Tubes ◽  
Protein Levels ◽  
Human Fallopian Tube

Heterogeneous nuclear ribonucleoproteins (hnRNPs), which are chromatin-associated RNA-binding proteins, participate in mRNA stability, transport, intracellular localization, and translation by acting as transacting factors. Several studies have shown that steroid hormones can regulate hnRNP expression. However, to date, the regulation of hnRNPs and their interactions with steroid hormone signaling in fallopian tubes and endometrium are not fully elucidated. In the present study, we determined whether hnRNP expression is regulated during the menstrual cycle and correlates with estrogen receptor (ER) and progesterone receptor (PR) levels in human fallopian tubes in vivo. Because of the limited availability of human tubal tissues for the research, we also explored the mechanisms of hnRNP regulation in human endometrium in vitro. Fallopian tissue was obtained from patients in the early, late, and postovulatory phases and the midsecretory phase and endometrial tissue from premenopausal and postmenopausal women undergoing hysterectomy. We measured expression of hnRNPs and assessed their intracellular localization and interactions with ERs and PRs. We also determined the effects of human chorionic gonadotropin, 17β-estradiol (E2), and progesterone (P4) on hnRNP expression. In fallopian tubes, mRNA and protein levels of hnRNP A1, AB, D, G, H, and U changed dynamically during ovulation and in the midsecretory phase. In coimmunolocation and coimmunoprecipitation experiments, hnRNPs interacted with each other and with ERs and PRs in fallopian tubes. After treatment with E2 and/or P4 to activate ERs and PRs, hnRNP A1, AB, D, G, and U proteins displayed overlapping but distinct patterns of regulation in the endometrium in vitro. Our findings expand the physiological repertoire of hnRNPs in human fallopian tubes and endometrium and suggest that steroid hormones regulate different hnRNPs directly by interacting with ERs and/or PRs or indirectly by binding other hnRNPs. Both actions may contribute to regulation of gene transcription.

scholarly journals The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277 ◽  
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

scholarly journals In vivo and in vitro arginine methylation of RNA-binding proteins.

1995 ◽  
Vol 15 (5) ◽  
pp. 2800-2808 ◽  
Author(s):  
Q Liu ◽  
G Dreyfuss
Keyword(s):  
Hela Cells ◽  
Posttranslational Modifications ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Arginine Methylation ◽  
Hnrnp A1 ◽  
Arginine Residues

Heterogenous nuclear ribonucleoproteins (hnRNPs) bind pre-mRNAs and facilitate their processing into mRNAs. Many of the hnRNPs undergo extensive posttranslational modifications including methylation on arginine residues. hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. The role of this modification is not known. Here we identify the hnRNPs that are methylated in HeLa cells and demonstrate that most of the pre-mRNA-binding proteins receive this modification. Using recombinant human hnRNP A1 as a substrate, we have partially purified and characterized a protein-arginine N-methyltransferase specific for hnRNPs from HeLa cells. This methyltransferase can methylate the same subset of hnRNPs in vitro as are methylated in vivo. Furthermore, it can also methylate other RNA-binding proteins that contain the RGG motif RNA-binding domain. This activity is evolutionarily conserved from lower eukaryotes to mammals, suggesting that methylation has a significant role in the function of RNA-binding proteins.

STREME: Accurate and versatile sequence motif discovery

2020 ◽  
Author(s):  
Timothy L. Bailey
Keyword(s):  
Motif Discovery ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Statistical Significance ◽  
Sequence Motif ◽  
Fully Integrated ◽  
Dna And Rna ◽  
Discovery Algorithms

AbstractSequence motif discovery algorithms can identify novel sequence patterns that perform biological functions in DNA, RNA and protein sequences—for example, the binding site motifs of DNA- and RNA-binding proteins. The STREME algorithm presented here advances the state-of-the-art in ab initio motif discovery in terms of both accuracy and versatility. Using in vivo DNA (ChIP-seq) and RNA (CLIP-seq) data, and validating motifs with reference motifs derived from in vitro data, we show that STREME is more accurate, sensitive, thorough and rapid than several widely used algorithms (DREME, HOMER, MEME, Peak-motifs and Weeder). STREME’s capabilities include the ability to find motifs in datasets with hundreds of thousands of sequences, to find both short and long motifs (from 3 to 30 positions), to perform differential motif discovery in pairs of sequence datasets, and to find motifs in sequences over virtually any alphabet (DNA, RNA, protein and user-defined alphabets). Unlike most motif discovery algorithms, STREME accurately estimates and reports the statistical significance of each motif that it discovers. STREME is easy to use via its web server at http://meme-suite.org, and is fully integrated with the widely-used MEME Suite of sequence analysis tools, which can be freely downloaded at the same web site for non-commercial use.

scholarly journals PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins

2018 ◽  
Author(s):  
Yongjia Duan ◽  
Aiying Du ◽  
Jinge Gu ◽  
Gang Duan ◽  
Chen Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Posttranslational Modifications ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Hnrnp A1 ◽  
Rnp Granules

SUMMARYMutations in RNA-binding proteins localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregations, which are pathological hallmarks in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of PARylation, an important PTM involved in DNA damage repair and cell death, in RNP-related neurodegeneration. We reveal that PARylation levels are a major regulator of the dynamic assembly-disassembly of RNP granules, and the disease-related RNPs such as hnRNP A1 and TDP-43 can both be PARylated and bind to PARylated proteins. We further identify the PARylation site of hnRNP A1 at K298, which controls the cytoplasmic translocation of hnRNP A1 in response to stress, as well as the PAR-binding motif (PBM) of hnRNP A1, which is required for the delivery and association of hnRNP A1 to stress granules. Moreover, we show that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, we establish that both genetic and pharmacological inhibition of PARP mitigates hnRNP A1 and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings indicate a novel and crucial role of PARylation in regulating the assembly and the dynamics of RNP granules, and dysregulation of PARylation may contribute to ALS disease pathogenesis.

The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

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