scholarly journals Splicing factor SRSF1 expands the regulatory logic of microRNA expression

2020 ◽  
Author(s):  
Marija Dargyte ◽  
Julia Philipp ◽  
Christina D. Palka ◽  
Michael D. Stone ◽  
Jeremy R. Sanford
Keyword(s):  
Binding Sites ◽  
Regulatory Elements ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Stem Loop ◽  
Microrna Biogenesis ◽  
Evolutionarily Conserved ◽  
Rna Interaction

AbstractThe serine and arginine-rich splicing factor SRSF1 is an evolutionarily conserved, essential pre-mRNA splicing factor. Through a global protein-RNA interaction survey we discovered SRSF1 binding sites 25-50nt upstream from hundreds of pre-miRNAs. Using primary miRNA-10b as a model we demonstrate that SRSF1 directly regulates microRNA biogenesis both in vitro and in vivo. Selective 2’ hydroxyl acylation analyzed by primer extension (SHAPE) defined a structured RNA element located upstream of the precursor miRNA-10b stem loop. Our data support a model where SRSF1 promotes initial steps of microRNA biogenesis by relieving the repressive effects of cis-regulatory elements within the leader sequence.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4349-4358 ◽  
Author(s):  
J. Charite ◽  
W. de Graaff ◽  
D. Consten ◽  
M.J. Reijnen ◽  
J. Korving ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Positional Information ◽  
Regulatory Elements ◽  
Gene Products ◽  
Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

scholarly journals Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 627-635 ◽  
Author(s):  
D.L. Song ◽  
G. Chalepakis ◽  
P. Gruss ◽  
A.L. Joyner
Keyword(s):  
Dna Binding ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Developing Brain ◽  
Molecular Evidence ◽  
Regulatory Sequences ◽  
Pax Genes

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

scholarly journals The MCP silencer of theDrosophila Abd-Bgene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

Development ◽  
2001 ◽  
Vol 128 (11) ◽  
pp. 2163-2173 ◽  
Author(s):  
Ana Busturia ◽  
Alan Lloyd ◽  
Fernando Bejarano ◽  
Michael Zavortink ◽  
Hua Xin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Homeotic Gene ◽  
Gaga Factor ◽  
Polycomb Response Elements ◽  
Silencer Element

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.

scholarly journals A Novel Splicing Factor, Yju2, Is Associated with NTC and Acts after Prp2 in Promoting the First Catalytic Reaction of Pre-mRNA Splicing

2007 ◽  
Vol 27 (15) ◽  
pp. 5403-5413 ◽  
Author(s):  
Yen-Chi Liu ◽  
Hsin-Chou Chen ◽  
Nan-Ying Wu ◽  
Soo-Chen Cheng
Keyword(s):  
Catalytic Reaction ◽  
Structural Rearrangement ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Complementation Analysis ◽  
Independent Manner ◽  
Heat Resistant ◽  
Spliceosome Activation

ABSTRACT The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.

scholarly journals HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

2017 ◽  
Author(s):  
Jonathan M. Howard ◽  
Hai Lin ◽  
Garam Kim ◽  
Jolene M Draper ◽  
Maximilian Haeussler ◽  
...  
Keyword(s):  
Splice Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Spliceosome Assembly ◽  
Over Expression

AbstractAlternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3’ splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3’ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3’ splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to “decoy” binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3’ splice sites.

scholarly journals Cooperative Pho2-Pho4 Interactions at thePHO5 Promoter Are Critical for Binding of Pho4 to UASp1 and for Efficient Transactivation by Pho4 at UASp2

1998 ◽  
Vol 18 (5) ◽  
pp. 2629-2639 ◽  
Author(s):  
Slobodan Barbaric ◽  
Martin Münsterkötter ◽  
Colin Goding ◽  
Wolfram Hörz
Keyword(s):  
Binding Sites ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Striking Difference ◽  
Homeodomain Protein ◽  
Interaction Domain ◽  
Independent Manner ◽  
Activity Measurements

ABSTRACT The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes toPHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

scholarly journals Evolutionarily Conserved Long Non-coding RNA Regulates Gene Expression in Cytokine Storm During COVID-19

2021 ◽  
Vol 8 ◽  
Author(s):  
Olanrewaju B. Morenikeji ◽  
Kahleel Bernard ◽  
Ellis Strutton ◽  
Madeleine Wallace ◽  
Bolaji N. Thomas
Keyword(s):  
Gene Expression ◽  
Drug Targets ◽  
Regulatory Elements ◽  
Cytokine Storm ◽  
Cellular Processes ◽  
Non Coding Rna ◽  
Evolutionarily Conserved ◽  
Multiple Species

Coronavirus is a family of viruses including alpha-, beta-, gamma-, delta-coronaviruses. Only alpha- and betacoronaviruses have been observed to infect humans. Past outbreaks of SARS-CoV and MERS-CoV, both betacoronavirus, are the result of a spillover from animals. Recently, a new strain termed SARS-CoV-2 emerged in December 2019 in Wuhan, China. Severe cases of COVID-19, the disease caused by SARS-CoV-2, lead to acute respiratory distress syndrome (ARDS). One contributor to the development of ARDS is cytokine storm, an overwhelming inflammatory immune response. Long non-coding RNAs (lncRNAs) are genetic regulatory elements that, among many functions, alter gene expression and cellular processes. lncRNAs identified to be pertinent in COVID-19 cytokine storm have the potential to serve as disease markers or drug targets. This project aims to computationally identify conserved lncRNAs potentially regulating gene expression in cytokine storm during COVID-19. We found 22 lncRNAs that can target 10 cytokines overexpressed in COVID-19 cytokine storm, 8 of which targeted two or more cytokine storm cytokines. In particular, the lncRNA non-coding RNA activated by DNA damage (NORAD), targeted five out of the ten identified cytokine storm cytokines, and is evolutionarily conserved across multiple species. These lncRNAs are ideal candidates for further in vitro and in vivo analysis.

scholarly journals Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009500
Author(s):  
Sabine Mohr ◽  
Andrew Kenny ◽  
Simon T. Y. Lam ◽  
Miles B. Morgan ◽  
Craig A. Smibert ◽  
...  
Keyword(s):  
Binding Sites ◽  
Inhibitory Effect ◽  
Multiple Roles ◽  
Rna Transport ◽  
Nurse Cells ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Dependent Inhibition

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3’ UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3’ UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.

Characterization of the Epstein–Barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1791-1799 ◽  
Author(s):  
Carine Segouffin-Cariou ◽  
Géraldine Farjot ◽  
Alain Sergeant ◽  
Henri Gruffat
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Regulatory Elements ◽  
Barr Virus ◽  
Viral Genes ◽  
Epstein Barr

The switch from latency to a productive cycle in Epstein–Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5′ end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions −469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.

A Stem-Loop in the 3′-UTR Mediates Iron-Dependent Regulation of Alpha Hemoglobin Stabilizing Protein mRNA Stability.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3628-3628
Author(s):  
Camila O. dos Santos ◽  
Adriana Duarte ◽  
Louis C. Dore ◽  
Sara Saad ◽  
Mitchell J. Weiss ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Regulatory Elements ◽  
In Vivo Studies ◽  
Beta Thalassemia ◽  
Binding Studies ◽  
Iron Starvation ◽  
Stem Loop ◽  
Alpha Globin

Abstract Alpha-hemoglobin stabilizing protein (AHSP) acts as a molecular chaperone that binds and stabilizes alpha globin, minimizing the formation of cytotoxic alpha-globin precipitates in red blood cells. It has been proposed that variations in AHSP protein levels could influence disease severity in patients with beta-thalassemia. A number of genomic sequences that could be involved with AHSP transcriptional regulation have been studied. Multiple consensus GATA-1 binding motifs and a single CACCC-binding site have been identified and characterized. In vivo studies demonstrated that AHSP gene transcription is dependent on both EKLF and GATA-1 transcription factors. Here, we identify an IRE (iron response-like element) in the AHSP 3′ untranslated region that is predicted to regulate mRNA stability. IREs are regulatory elements that encode a phylogenetically defined sequence-structure motif that provide a specific recognization site for the IRE-binding proteins (IRE-BP1 or IRE-BP2). Iron starvation of cells induces high affinity binding of cytoplasmic IRE-BP to an IRE, which has at least two distinct known biological consequences. IRE in the 5′-UTR of an mRNA provides for iron-dependent regulation of translation, whereas an IRE in the 3′-UTR confers iron-dependent regulation of mRNA stability. Computer modeling identified a potential IRE stem-loop structure near the 3′-end of murine and human AHSP mRNAs. In vitro binding studies indicate that this structure recognizes an IRE-BP. Moreover, the affinity of this interaction was reduced by mutations that disrupt base pairing within the stem-loop. Iron supplementation, which is predicted to decrease IRE-BP affinity for the IRE, destabilizes AHSP in human and mouse erythroleukemia cells. These results indicate that AHSP expression is regulated by RNA stability in an iron-dependent fashion. Our findings have implications for the control of AHSP expression in beta-thalassemia.

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