scholarly journals Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

2021 ◽  
Author(s):  
Giulia Biancon ◽  
Poorval Joshi ◽  
Joshua T Zimmer ◽  
Torben Hunck ◽  
Yimeng Gao ◽  
...  
Keyword(s):  
Rna Binding ◽  
Specific Binding ◽  
Aberrant Splicing ◽  
Binding Model ◽  
Full Spectrum ◽  
Myeloid Malignancies ◽  
Rna Granules ◽  
Rna Biology ◽  
Site Recognition

AbstractSomatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3’ splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

scholarly journals The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

2004 ◽  
Vol 24 (10) ◽  
pp. 4229-4240 ◽  
Author(s):  
Christopher J. Webb ◽  
Jo Ann Wise
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Large Subunit ◽  
Time Frame ◽  
Small Subunit ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Binding Domains ◽  
Site Recognition

ABSTRACT The small subunit of U2AF, which functions in 3′ splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association

2021 ◽  
Author(s):  
Mina N. Anadolu ◽  
Senthilkumar Kailasam ◽  
Konstanze Simbriger ◽  
Jingyu Sun ◽  
Teodora Markova ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Cytoskeletal Proteins ◽  
Rna Granules ◽  
Brain Extracts ◽  
The Brain

AbstractLocal translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched within neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3’end and were found in reproducible peaks in the mRNAs. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules.Significance StatementThis work finds that neuronal ribosomes in RNA granules are concentrated at consensus sites previously identified through cross-linking FMRP to mRNAs in the brain. This strongly links the compacted ribosomes found in the pellet of sucrose gradients from brain extracts to stalled ribosomes regulated by FMRP and provides important insights into how stalling is accomplished. Many mRNAs important for neurodevelopment are enriched in these ribosomes. These results suggest that many studies on translation in the brain may need to be revised. The larger size of the ribosomal footprints on stalled polysomes and their sedimentation in the pellet of sucrose gradients suggests mRNAs found in these structures have not been assessed in many studies of neuronal translation.

scholarly journals Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jarrett Smith ◽  
Deepika Calidas ◽  
Helen Schmidt ◽  
Tu Lu ◽  
Dominique Rasoloson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Intrinsically Disordered Protein ◽  
General Mechanism ◽  
P Granules ◽  
Rna Granules ◽  
Intrinsically Disordered ◽  
Disordered Protein

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2019 ◽  
Author(s):  
Erin Cutts ◽  
J. Barry Egan ◽  
Ian Dodd ◽  
Keith Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

AbstractThe Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl’s repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals The RNA-binding protein GRSF1 promotes hepatocarcinogenesis via competitively binding to YY1 mRNA with miR-30e-5p

2022 ◽  
Vol 41 (1) ◽  
Author(s):  
Lili Han ◽  
Chen Huang ◽  
Xiaofei Wang ◽  
Dongdong Tong
Keyword(s):  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Xenograft Model ◽  
Interaction Network ◽  
Rna Immunoprecipitation ◽  
Small Molecule Compound ◽  
Underlying Mechanisms

Abstract Background Dysregulation of RNA binding protein (RBP) expression has been confirmed to be causally linked with tumorigenesis. The detailed biological effect and underlying mechanisms of the RBP GRSF1 in hepatocellular carcinoma (HCC) remain unclear. Methods HCC cells with stable knockdown of GRSF1 were established using two sh-RNA-encoding lentiviruses. The functions of GRSF1 in HCC were explored using MTT, colony formation, flow cytometry, and Transwell assays and a xenograft model. Transcriptomic sequencing in GRSF1-deficient MHCC-97H cells was carried out to identify the downstream effector of GRSF1. The regulatory mechanisms among GRSF1, YY1 and miR-30e-5p were investigated via RNA immunoprecipitation, luciferase, RNA pull-down and ChIP assays. Several in vivo assays were used to assess the selectivity of the small-molecule compound VE-821 in HCC and to confirm the absence of general toxicity in animal models. Results GRSF1 was frequently increased in HCC tissue and cells and was associated with worse clinical outcomes. GRSF1 functions as a novel oncogenic RBP by enhancing YY1 mRNA stability, and the GUUU motifs within the YY1 3`UTR 2663-2847 were the specific binding motifs for GRSF1. YY1 feedback promoted GRSF1 expression by binding to the GRSF1 promoter. In addition, YY1 was a critical target of miR-30e-5p, which was confirmed in this study to inhibit HCC hepatocarcinogenesis. GRSF1 and miR-30e-5p competitively regulated YY1 by binding to its 3`UTR 2663-2847 region. Finally, we identified that VE-821 blocked HCC progression by inhibiting the GRSF1/YY1 pathway. Conclusion This study revealed the interaction network among GRSF1, YY1 and miR-30e-5p, providing new insight into HCC pathogenesis, and indicated that VE821 may serve as a novel agent with potential for HCC treatment through inhibition of the GRSF1/YY1 axis.

scholarly journals The Rna-binding Protein GRSF1 Promote shepatocarcinogenesis via competitively Binding Toyy1 mRNA with mIR-30e-5p

2020 ◽  
Author(s):  
Lili Han ◽  
Xiaofei Wang ◽  
Fei Wu ◽  
Chen Huang ◽  
Shuqun Zhang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Human Cancer ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Xenograft Model ◽  
Interaction Network ◽  
Luciferase Assay

Abstract Background:Numerous studies have highlighted that dysregulation of RNA binding protein (RBP) expression is causally linked with human cancer tumorigenesis. The detailed biological effect and underlying mechanisms of RBP GRSF1 in hepatocellular carcinoma (HCC) remain unclear.Methods:GRSF1 expression in HCC tissues and cells was measured by western blotting and qRT-PCR assays. HCC cells with stable knockdown of GRSF1 were established using two sh-RNA encoding lentiviruses. Then the functions of GRSF1 in HCC were explored using MTT, colony formation, flow cytometry, Transwell assays and xenograft model. Transcriptometric sequencing in GRSF1-deficient MHCC-97H cells were carried out to identify the downstream effector of GSRF1. The regulatory mechanisms among GRSF1, YY1 and miR-30e-5p were investigated by RNA immunoprecipitation, luciferase assay, RNA pull down and ChIP assay.Results:GRSF1was frequently increased in HCC tissue and cells with a worse clinical outcomes in HCC. GRSF1 worked as a novel oncogenic RBP in vitro and in vivo via post-regulating the stability of YY1 mRNA. And the GUUU motifs on YY1 3`UTR 2663-2847 was the specific binding motifs for GRSF1. More interesting, YY1 feedback promoted GRSF1 expression by binding to GRSF1 promoters. In addition, YY1 was a critical target of miR-30e-5p, which was confirmed by our data that inhibited HCC hepatocarcinogenesis. Further investigation showed GRSF1 and miR-30e-5p competitively regulated YY1 via binding to its YY1 3`UTR 2663-2847 region. At last, we identified that 3-amino-6-(4-methylsulfonylphenyl)-N-phenylpyrazine-2-carboxamide (VE821) blocked HCC progression by inhibiting the GRSF1/YY1 pathway.Conclusions:GRSF1 promoted hepatocarcinogenesis via competitively binding to YY1 3`UTR 2663-2847 with miR-30e-5p.The interaction network among GRSF1, YY1 and miR-30e-5p provided a new insight for the HCC pathogenesis, and VE821 may serve as a novel agent with potential for HCC treatment via inhibiting GRSF1/YY1 axis.

scholarly journals Discovering the RNA-Binding Proteome of Plant Leaves with an Improved RNA Interactome Capture Method

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 661 ◽  
Author(s):  
Marcel Bach-Pages ◽  
Felix Homma ◽  
Jiorgos Kourelis ◽  
Farnusch Kaschani ◽  
Shabaz Mohammed ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Leaf Tissue ◽  
Plant Leaves ◽  
Binding Domains ◽  
Rna Biology ◽  
Full Complement ◽  
Leaf Tissues ◽  
Capture Method

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues.

scholarly journals RNA Granules: A View from the RNA Perspective

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3130 ◽  
Author(s):  
Siran Tian ◽  
Harrison A. Curnutte ◽  
Tatjana Trcek
Keyword(s):  
Transcriptional Regulation ◽  
Rna Structure ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Special Issue ◽  
Rna Granules ◽  
Rna Biology ◽  
A Cell ◽  
Post Transcriptional Regulation

RNA granules are ubiquitous. Composed of RNA-binding proteins and RNAs, they provide functional compartmentalization within cells. They are inextricably linked with RNA biology and as such are often referred to as the hubs for post-transcriptional regulation. Much of the attention has been given to the proteins that form these condensates and thus many fundamental questions about the biology of RNA granules remain poorly understood: How and which RNAs enrich in RNA granules, how are transcripts regulated in them, and how do granule-enriched mRNAs shape the biology of a cell? In this review, we discuss the imaging, genetic, and biochemical data, which have revealed that some aspects of the RNA biology within granules are carried out by the RNA itself rather than the granule proteins. Interestingly, the RNA structure has emerged as an important feature in the post-transcriptional control of granule transcripts. This review is part of the Special Issue in the Frontiers in RNA structure in the journal Molecules.

scholarly journals Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

2016 ◽  
Author(s):  
Jarrett Smith ◽  
Deepika Calidas ◽  
Helen Schmidt ◽  
Tu Lu ◽  
Dominique Rasoloson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Intrinsically Disordered Protein ◽  
P Granules ◽  
C Elegans ◽  
Rna Granules ◽  
Intrinsically Disordered ◽  
Disordered Protein

ABSTRACTRNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings support a model whereby MEX-5 functions as an mRNA sink to locally suppress MEG-3 phase separation and drive P granule asymmetry.HIGHLIGHTS- The intrinsically-disordered protein MEG-3 is essential for localized assembly of P granules in C. elegans zygotes.- MEG-3 binds RNA and RNA stimulates MEG-3 phase separation.- The RNA-binding protein MEX-5 inhibits MEG-3 granule assembly in the anterior cytoplasm by sequestering RNA.

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