scholarly journals Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing

2018 ◽  
Author(s):  
Kaushik Saha ◽  
Whitney England ◽  
Mike Minh Fernandez ◽  
Tapan Biswas ◽  
Robert C. Spitale ◽  
...  
Keyword(s):  
Structural Information ◽  
Structural Features ◽  
Splicing Factors ◽  
Splice Sites ◽  
Exonic Splicing Enhancer ◽  
Stem Loop ◽  
The Core ◽  
Retained Introns ◽  
Splicing Enhancer ◽  
Structural Disruption

ABSTRACTRecognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

scholarly journals Structural disruption of exonic stem–loops immediately upstream of the intron regulates mammalian splicing

2020 ◽  
Vol 48 (11) ◽  
pp. 6294-6309 ◽  
Author(s):  
Kaushik Saha ◽  
Whitney England ◽  
Mike Minh Fernandez ◽  
Tapan Biswas ◽  
Robert C Spitale ◽  
...  
Keyword(s):  
Structural Information ◽  
Structural Features ◽  
Splicing Factors ◽  
Splice Sites ◽  
Exonic Splicing Enhancer ◽  
Stem Loop ◽  
The Core ◽  
Retained Introns ◽  
Splicing Enhancer ◽  
Structural Disruption

Abstract Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem–loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

scholarly journals A systems view of spliceosomal assembly and branchpoints with iCLIP

2018 ◽  
Author(s):  
Michael Briese ◽  
Nejc Haberman ◽  
Christopher R. Sibley ◽  
Anob M. Chakrabarti ◽  
Zhen Wang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Splicing Factors ◽  
Splice Sites ◽  
Dynamic Nature ◽  
The Core ◽  
Spliceosomal Protein ◽  
Systems View ◽  
Transcriptomic Studies

AbstractStudies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map human spliceosome engagement with snRNAs and pre-mRNAs. This identified over 50,000 branchpoints (BPs) that have canonical sequence and structural features. Moreover, it revealed 7 binding peaks around BPs and splice sites, each precisely overlapping with binding profiles of specific splicing factors. We show how the binding patterns of these RBPs are affected by the position and strength of BPs. For example, strong or proximally located BPs preferentially bind SF3 rather than U2AF complex. Notably, these effects are partly neutralized during spliceosomal assembly in a way that depends on the core spliceosomal protein PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

scholarly journals The doublesex Splicing Enhancer Components Tra2 and Rbp1 Also Repress Splicing through an Intronic Silencer

2006 ◽  
Vol 27 (2) ◽  
pp. 699-708 ◽  
Author(s):  
Junlin Qi ◽  
Shihuang Su ◽  
William Mattox
Keyword(s):  
Branch Point ◽  
Binding Sites ◽  
Splicing Factors ◽  
Splice Sites ◽  
Consensus Binding Site ◽  
Synergistic Interactions ◽  
Splicing Silencer ◽  
Specific Alternative ◽  
Splicing Enhancer

ABSTRACT The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

scholarly journals Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

1996 ◽  
Vol 16 (5) ◽  
pp. 2325-2331 ◽  
Author(s):  
R R Gontarek ◽  
D Derse
Keyword(s):  
Alternative Splicing ◽  
Exon Skipping ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Exonic Splicing Enhancer ◽  
Exon Inclusion ◽  
Alternative Mrna Splicing ◽  
Splicing Enhancer

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

scholarly journals Evidence for Substrate-Specific Requirement of the Splicing Factor U2AF35 and for Its Function after Polypyrimidine Tract Recognition by U2AF65

1999 ◽  
Vol 19 (12) ◽  
pp. 8263-8271 ◽  
Author(s):  
Sabine Guth ◽  
Concepción Martínez ◽  
Rajesh K. Gaur ◽  
Juan Valcárcel
Keyword(s):  
Protein Interactions ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Polypyrimidine Tract ◽  
Protein Protein Interactions ◽  
Exonic Splicing Enhancer ◽  
U2 Snrnp ◽  
And Function ◽  
Auxiliary Factor ◽  
Splicing Enhancer

ABSTRACT U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF65 and U2AF35. U2AF65 binds to the polypyrimidine (Py) tract upstream from the 3′ splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF35 facilitates U2AF65 binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF35 remain controversial. Here we show that recombinant U2AF65 is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF65, U2AF35, and the interaction between them are required for splicing of an immunoglobulin μ pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF35 occurs without changes in U2AF65cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF35 and a novel function for this factor in pre-mRNA splicing.

Measurement and Reduction of Radiation Damage in Frozen Hydrated Crystalline Specimens

1978 ◽  
Vol 36 (3) ◽  
pp. 70-77 ◽  
Author(s):  
R.M. Glaeser ◽  
S.B. Hayward
Keyword(s):  
Diffraction Pattern ◽  
Structural Information ◽  
Structural Disorder ◽  
Structural Features ◽  
Biological Macromolecules ◽  
Diffraction Intensity ◽  
Object Structure ◽  
Specimen Material ◽  
Unit Cells ◽  
Identical Unit

Highly ordered or crystalline biological macromolecules become severely damaged and structurally disordered after a brief electron exposure. Evidence that damage and structural disorder are occurring is clearly given by the fading and eventual disappearance of the specimen's electron diffraction pattern. The fading and disappearance of sharp diffraction spots implies a corresponding disappearance of periodic structural features in the specimen. By the same token, there is a oneto- one correspondence between the disappearance of the crystalline diffraction pattern and the disappearance of reproducible structural information that can be observed in the images of identical unit cells of the object structure. The electron exposures that result in a significant decrease in the diffraction intensity will depend somewhat upon the resolution (Bragg spacing) involved, and can vary considerably with the chemical makeup and composition of the specimen material.

HAZARD: An Expert System for Risk Assessment of Environmental Chemicals

1987 ◽  
Vol 26 (01) ◽  
pp. 13-23 ◽  
Author(s):  
H. W. Gottinger
Keyword(s):  
Expert System ◽  
Structural Information ◽  
Chemical Carcinogenesis ◽  
Structural Features ◽  
Environmental Chemicals ◽  
Chemical Carcinogens ◽  
Carcinogenic Activity ◽  
Current State ◽  
Structure Activity ◽  
Carcinogenic Potential

AbstractThe purpose of this paper is to report on an expert system in design that screens for potential hazards from environmental chemicals on the basis of structure-activity relationships in the study of chemical carcinogenesis, particularly with respect to analyzing the current state of known structural information about chemical carcinogens and predicting the possible carcinogenicity of untested chemicals. The structure-activity tree serves as an index of known chemical structure features associated with carcinogenic activity. The basic units of the tree are the principal recognized classes of chemical carcinogens that are subdivided into subclasses known as nodes according to specific structural features that may reflect differences in carcinogenic potential among chemicals in the class. An analysis of a computerized data base of known carcinogens (knowledge base) is proposed using the structure-activity tree in order to test the validity of the tree as a classification scheme (inference engine).

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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