scholarly journals Splicing at the phase-separated nuclear speckle interface: a model

2020 ◽  
Author(s):  
Susan E Liao ◽  
Oded Regev
Keyword(s):  
Phase Separation ◽  
Rna Splicing ◽  
Splicing Factors ◽  
Sequence Motifs ◽  
Splice Sites ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Functional Roles ◽  
Hnrnp Proteins ◽  
Model Sequence

Abstract Phase-separated membraneless bodies play important roles in nucleic acid biology. While current models for the roles of phase separation largely focus on the compartmentalization of constituent proteins, we reason that other properties of phase separation may play functional roles. Specifically, we propose that interfaces of phase-separated membraneless bodies could have functional roles in spatially organizing biochemical reactions. Here we propose such a model for the nuclear speckle, a membraneless body implicated in RNA splicing. In our model, sequence-dependent RNA positioning along the nuclear speckle interface coordinates RNA splicing. Our model asserts that exons are preferentially sequestered into nuclear speckles through binding by SR proteins, while introns are excluded through binding by nucleoplasmic hnRNP proteins. As a result, splice sites at exon-intron boundaries are preferentially positioned at nuclear speckle interfaces. This positioning exposes splice sites to interface-localized spliceosomes, enabling the subsequent splicing reaction. Our model provides a simple mechanism that seamlessly explains much of the complex logic of splicing. This logic includes experimental results such as the antagonistic duality between splicing factors, the position dependence of splicing sequence motifs, and the collective contribution of many motifs to splicing decisions. Similar functional roles for phase-separated interfaces may exist for other membraneless bodies.

scholarly journals RNAs as proximity labeling media for identifying nuclear speckle positions relative to the genome

2018 ◽  
Author(s):  
Weizhong Chen ◽  
Zhangming Yan ◽  
Simin Li ◽  
Norman Huang ◽  
Xuerui Huang ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Genomic Sequence ◽  
Single Cells ◽  
Regulation Of Gene Expression ◽  
Nuclear Genome ◽  
Fold Increase ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Interaction Sites ◽  
Genomic Regions

AbstractNuclear speckles are interchromatin structures enriched in RNA splicing factors. Determining their relative positions with respect to the folded nuclear genome could provide critical information on co-and post-transcriptional regulation of gene expression. However, it remains challenging to identify which parts of the nuclear genome are in proximity to nuclear speckles, due to physical separation between nuclear speckle cores and chromatin. We hypothesized that noncoding RNAs including small nuclear RNAs, 7SK and Malat1, which accumulate at the periphery of nuclear speckles (nsaRNA,nuclearspeckleassociated RNA), may extend to sufficient proximity to the nuclear genome. Leveraging a transcriptome-genome interaction assay (MARGI), we identified nsaRNA-interacting genomic sequences, which exhibited clustering patterns (nsaPeaks) in the genome, suggesting existence of relatively stable interaction sites for nsaRNAs in nuclear genome. Posttranscriptional pre-mRNAs, which are known to be clustered to nuclear speckles, exhibited proximity to nsaPeaks but rarely to other genomic regions. Furthermore, CDK9 proteins that localize to the vicinity of nuclear speckles produced ChIP-seq peaks that overlapped with nsaPeaks. Our combined DNA FISH and immunofluorescence analysis in 182 single cells revealed a 3-fold increase in odds for nuclear speckles to localize near an nsaPeak than its neighboring genomic sequence. These data suggest a model that nsaRNAs locate in sufficient proximity to nuclear genome and leave identifiable genomic footprints, thus revealing the parts of genome proximal to nuclear speckles.

scholarly journals Functional roles of protein splicing factors

2012 ◽  
Vol 32 (4) ◽  
pp. 345-359 ◽  
Author(s):  
Hsin-Chou Chen ◽  
Soo-Chen Cheng
Keyword(s):  
Rna Splicing ◽  
Structural Changes ◽  
Dynamic Structure ◽  
Splicing Factors ◽  
Protein Splicing ◽  
Rna Helicases ◽  
Functional Roles ◽  
Assembly Pathway ◽  
Small Nuclear Rnas ◽  
Sequential Binding

RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors. DExD/H-box RNA helicases are required to mediate structural changes in the spliceosome at various steps in the assembly pathway and have also been implicated in the fidelity control of the splicing reaction. Other proteins also play key roles in mediating the progression of the spliceosome pathway. In this review, we discuss the functional roles of the protein factors involved in the spliceosome pathway primarily from studies in the yeast system.

scholarly journals Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

2005 ◽  
Vol 25 (21) ◽  
pp. 9586-9594 ◽  
Author(s):  
T. Nicolai Siegel ◽  
Kevin S. W. Tan ◽  
George A. M. Cross
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Splicing ◽  
Open Reading Frames ◽  
Sequence Motifs ◽  
Branch Points ◽  
Splice Sites ◽  
Reporter System ◽  
Specific Sequence ◽  
Mrna Maturation ◽  
Splicing Efficiency

ABSTRACT mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5′ splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3′ splice sites (3′SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3′SS, we constructed a luciferase-β-galactosidase double-reporter system. By testing ∼90 sequences, we demonstrated that the optimum poly(Y) tract length is ∼25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3′SS is important, and an AC dinucleotide at positions −3 and −4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3′SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.

scholarly journals USP42 drives nuclear speckle mRNA splicing via directing dynamic phase separation to promote tumorigenesis

2021 ◽  
Author(s):  
Shuyan Liu ◽  
Taishu Wang ◽  
Yulin Shi ◽  
Lu Bai ◽  
Shanshan Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Posttranslational Modifications ◽  
Bioinformatic Analysis ◽  
Mrna Splicing ◽  
Dependent Manner ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Dynamic Regulation ◽  
Intrinsically Disordered ◽  
The Impact

AbstractLiquid–liquid phase separation is considered a generic approach to organize membrane-less compartments, enabling the dynamic regulation of phase-separated assemblies to be investigated and pivotal roles of protein posttranslational modifications to be demonstrated. By surveying the subcellular localizations of human deubiquitylases, USP42 was identified to form nuclear punctate structures that are associated with phase separation properties. Bioinformatic analysis demonstrated that the USP42 C-terminal sequence was intrinsically disordered, which was further experimentally confirmed to confer phase separation features. USP42 is distributed to SC35-positive nuclear speckles in a positively charged C-terminal residue- and enzymatic activity-dependent manner. Notably, USP42 directs the integration of the spliceosome component PLRG1 into nuclear speckles, and its depletion interferes with the conformation of SC35 foci. Functionally, USP42 downregulation deregulates multiple mRNA splicing events and leads to deterred cancer cell growth, which is consistent with the impact of PLRG1 repression. Finally, USP42 expression is strongly correlated with that of PLRG1 in non-small-cell lung cancer samples and predicts adverse prognosis in overall survival. As a deubiquitylase capable of dynamically guiding nuclear speckle phase separation and mRNA splicing, USP42 inhibition presents a novel anticancer strategy by targeting phase separation.

scholarly journals SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles

2012 ◽  
Vol 23 (18) ◽  
pp. 3694-3706 ◽  
Author(s):  
Vidisha Tripathi ◽  
David Y. Song ◽  
Xinying Zong ◽  
Sergey P. Shevtsov ◽  
Stephen Hearn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Cell Nucleus ◽  
Interphase Nucleus ◽  
Sr Proteins ◽  
Mrna Processing ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Processing Factors

The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II–mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.

scholarly journals Targeted Quantification of Detergent-Insoluble RNA-Binding Proteins in Human Brain Reveals Stage and Disease Specific Co-aggregation in Alzheimer’s Disease

2021 ◽  
Vol 14 ◽  
Author(s):  
Qi Guo ◽  
Eric B. Dammer ◽  
Maotian Zhou ◽  
Sean R. Kundinger ◽  
Marla Gearing ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Rna Splicing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Spliceosome Assembly ◽  
Network Analyses

Core spliceosome and related RNA-binding proteins aggregate in Alzheimer’s disease (AD) brain even in early asymptomatic stages (AsymAD) of disease. To assess the specificity of RNA-binding protein aggregation in AD, we developed a targeted mass spectrometry approach to quantify broad classes of RNA-binding proteins with other pathological proteins including tau and amyloid beta (Aβ) in detergent insoluble fractions from control, AsymAD, AD and Parkinson’s disease (PD) brain. Relative levels of specific insoluble RNA-binding proteins across different disease groups correlated with accumulation of Aβ and tau aggregates. RNA-binding proteins, including splicing factors with homology to the basic-acidic dipeptide repeats of U1-70K, preferentially aggregated in AsymAD and AD. In contrast, PD brain aggregates were relatively depleted of many RNA-binding proteins compared to AsymAD and AD groups. Correlation network analyses resolved 29 distinct modules of co-aggregating proteins including modules linked to spliceosome assembly, nuclear speckles and RNA splicing. Modules related to spliceosome assembly and nuclear speckles showed stage-specific enrichment of insoluble RBPs from AsymAD and AD brains, whereas the RNA splicing module was reduced specifically in PD. Collectively, this work identifies classes of RNA-binding proteins that distinctly co-aggregate in detergent-insoluble fractions across the specific neurodegenerative diseases we examined.

scholarly journals C3G dynamically associates with Nuclear speckles and regulates mRNA splicing

2017 ◽  
Author(s):  
Dhruv Kumar Shakyawar ◽  
Bhattiprolu Muralikrishna ◽  
Vegesna Radha
Keyword(s):  
Rna Splicing ◽  
Mrna Splicing ◽  
C2c12 Cells ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Protein Levels ◽  
Ras Family ◽  
Reversible Association ◽  
And Function ◽  
Mammalian Embryonic Development

AbstractC3G (RapGEF1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleo-cytoplasmic exchange. Here we show that C3G localizes to SC35 positive nuclear speckles, and regulates splicing activity. Reversible association of C3G with speckles was seen upon inhibition of transcription and splicing. C3G shows partial colocalization with SC35, and is recruited to a chromatin and RNase sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton, and is lost upon expression of the kinase, Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles and inactivation of Rap signalling by expression of GFP- Rap1GAP, alters speckle morphology and number. Enhanced association of C3G with speckles is seen upon GSK3β inhibition, or differentiation of C2C12 cells to myotubes. CRISPR/Cas9 mediated knockdown of C3G resulted in decreased splicing activity and reduced staining for SC35 in speckles. C3G knockout clones of C2C12 as well as MDA-MB- 231 showed reduced protein levels of several splicing factors compared to control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.SummaryNuclear speckles are sites for pre-mRNA splicing. We provide evidence for localization and function of a Ras family GTPase, Rap1 and its exchange factor C3G in nuclear speckles.

Computational Approaches to Predict the Non-canonical DNAs

2019 ◽  
Vol 14 (6) ◽  
pp. 470-479 ◽  
Author(s):  
Nazia Parveen ◽  
Amen Shamim ◽  
Seunghee Cho ◽  
Kyeong Kyu Kim
Keyword(s):  
Computational Methods ◽  
Genetic Instability ◽  
Computational Prediction ◽  
Structure And Function ◽  
Sequence Motifs ◽  
Computational Approaches ◽  
Functional Roles ◽  
And Function ◽  
Genetic Events ◽  
Insight Into

Background: Although most nucleotides in the genome form canonical double-stranded B-DNA, many repeated sequences transiently present as non-canonical conformations (non-B DNA) such as triplexes, quadruplexes, Z-DNA, cruciforms, and slipped/hairpins. Those noncanonical DNAs (ncDNAs) are not only associated with many genetic events such as replication, transcription, and recombination, but are also related to the genetic instability that results in the predisposition to disease. Due to the crucial roles of ncDNAs in cellular and genetic functions, various computational methods have been implemented to predict sequence motifs that generate ncDNA. Objective: Here, we review strategies for the identification of ncDNA motifs across the whole genome, which is necessary for further understanding and investigation of the structure and function of ncDNAs. Conclusion: There is a great demand for computational prediction of non-canonical DNAs that play key functional roles in gene expression and genome biology. In this study, we review the currently available computational methods for predicting the non-canonical DNAs in the genome. Current studies not only provide an insight into the computational methods for predicting the secondary structures of DNA but also increase our understanding of the roles of non-canonical DNA in the genome.

scholarly journals Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kristin A. Ham ◽  
Niall P. Keegan ◽  
Craig S. McIntosh ◽  
May T. Aung-Htut ◽  
Khine Zaw ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Antisense Oligonucleotides ◽  
Live Cells ◽  
Splice Sites ◽  
Cryptic Splice Site ◽  
Exon Definition ◽  
Rna Targets ◽  
Cryptic Splice Sites ◽  
Six Genes ◽  
Intended Effect

AbstractAntisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

scholarly journals Regulation of RNA Splicing: Aberrant Splicing Regulation and Therapeutic Targets in Cancer

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 923
Author(s):  
Koji Kitamura ◽  
Keisuke Nimura
Keyword(s):  
Dna Sequences ◽  
Rna Splicing ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Aberrant Splicing ◽  
Aberrant Expression ◽  
Non Coding Rnas ◽  
Precursor Mrna ◽  
Splicing Enhancer ◽  
Small Nuclear Ribonucleoproteins

RNA splicing is a critical step in the maturation of precursor mRNA (pre-mRNA) by removing introns and exons. The combination of inclusion and exclusion of introns and exons in pre-mRNA can generate vast diversity in mature mRNA from a limited number of genes. Cancer cells acquire cancer-specific mechanisms through aberrant splicing regulation to acquire resistance to treatment and to promote malignancy. Splicing regulation involves many factors, such as proteins, non-coding RNAs, and DNA sequences at many steps. Thus, the dysregulation of splicing is caused by many factors, including mutations in RNA splicing factors, aberrant expression levels of RNA splicing factors, small nuclear ribonucleoproteins biogenesis, mutations in snRNA, or genomic sequences that are involved in the regulation of splicing, such as 5’ and 3’ splice sites, branch point site, splicing enhancer/silencer, and changes in the chromatin status that affect the splicing profile. This review focuses on the dysregulation of RNA splicing related to cancer and the associated therapeutic methods.

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