Multiple intron motifs have redundant functions in trans-splicing of the mod(mdg4) locus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1289078 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:In contrast to canonical cis-splicing, trans-splicing combines exons from two distinct transcripts, yielding chimeric mRNAs. One striking example is the ubiquitously expressed modifier of mdg4 (mod(mdg4)) locus in Drosophila, where all mRNAs spanning over 30 isoforms are generated exclusively by trans-splicing. This process combines common constitutive exons with one of many alternative 3′ variable exons transcribed from independent promoters. Here, we demonstrate that trans-splicing is determined by an approximately 600-bp region located in the proximal part of the last common intron. Deletion analysis in a model system and directly within the endogenous mod(mdg4) locus revealed that this intronic region contains multiple redundant motifs for RNA-binding proteins (RBPs), which collectively determine the level of trans-splicing. By affinity purification of the in vitro transcribed proximal part of the intronic RNA, we identified specific binding of a large group of spliceosome-associated RBPs, some of which were found to have binding motifs in the intronic region of mod(mdg4) RNA, which is critical for trans-splicing.
2026-05-28 | PXD079014 | Pride
Project description:In contrast to canonical cis-splicing, trans-splicing combines exons from two distinct transcripts, yielding chimeric mRNAs. This process is typically suppressed through the tight coupling of pre-mRNA synthesis and splicing, primarily due to the inherent risk of generating chimeric proteins with aberrant or deleterious properties. Nevertheless, trans-splicing permits the creation of a substantially greater diversity of mRNA isoforms that encode variants of a single protein. One compelling example is the mod(mdg4) locus in Drosophila, where all mRNAs, encompassing over 30 isoforms, are exclusively generated via trans-splicing, which integrates common constitutive exons with one of several alternative 3′ variable exons transcribed from independent promoters. This investigation analyzed the roles of the mod(mdg4) gene’s promoter, exons, and introns in trans-splicing. Our methodology involved a previously validated model in the heterogeneous 22A genomic region, augmented by targeted deletions within the fourth intron of the endogenous locus. We determined that the mod(mdg4) promoter only modestly enhances trans-splicing efficiency. Critically, the predominant determinants are multiple, redundant RNA-binding protein motifs concentrated within the proximal 412 bp of the fourth intron. These results collectively support a model wherein trans-splicing is primarily mediated by redundant intronic motifs recognized by spliceosome components.
2025-07-11 | GSE302152 | GEO
Project description:Here we map the localization of Mod(mdg4), including Mod(mdg4)2.2 specific and Mod(mdg4) common to all isoforms, to chromatin insulators, as well as the lethal-3 malignant brain tumor protein in Drosophila Kc cells.
2012-06-18 | GSE36393 | GEO
Project description:Here we map the localization of Mod(mdg4), including Mod(mdg4)2.2 specific and Mod(mdg4) common to all isoforms, to chromatin insulators, as well as the lethal-3 malignant brain tumor protein in Drosophila Kc cells. examination of genomic occupancy for Mod(mdg4)2.2, Mod(mdg4)BTB (all isoforms), and L(3)mbt, control samples used for Drosophila Kc were previously described (Wood, Van Bortle et al., 2011 GSE30740)
2012-06-18 | E-GEOD-36393 | biostudies-arrayexpress
Project description:Telomeres in Drosophila are composed of sequential non-LTR retrotransposons: Het-A, TART, TAHRE. Although their expression is highly dynamic, molecular mechanism governing these retrotransposons is poorly understood. Here, we show that specific splice variants of Mod(mdg4) act as repressors of Het-A by blocking subtelomeric enhancers in ovarian somatic cells. We found that a variant, Mod(mdg4)-N, can repress Het-A expression most efficiently among variants. Mod(mdg4)-N mutant flies show elevated Het-A expression and female sterility. Further investigation revealed that Mod(mdg4)-N-binding subtelomeric sequences exhibit enhancer-blocking activity, and recruitment of RNA polymerase II (Pol II) on subtelomeres by Mod(mdg4)-N is essential for this enhancer-blocking. Moreover, Mod(mdg4)-N functions to form chromatin boundaries of higher-order chromatin conformation but this mechanism is independent with its Pol II recruitment activity observed at telomeres/subtelomeres. This study provides an unexpected link between enhancer-blocking and telomere regulation, and two different molecular mechanisms exhibited by an insulator protein to orchestrate precise gene expression.
2022-10-20 | GSE176196 | GEO
Project description:Fly strains were obtained from the Bloomington Stock Center. The chromosomes containing the gene deletion mod(mdg4)L3101 {y[1] w[1118]; P{w[+mC]=lacW}mod(mdg4)[L3101]/TM3, Ser[1]}, HP1 {In(1)w[m4h]; Su(var)205[5]/In(2L)Cy,In(2R)Cy, Cy[1]}, mod(mdg4)G16853 {w[1118]; P{w[+mC]=EP}mod(mdg4)[G16853]/TM6C, Sb[1]}, and Jil-1Scim {y[1]; P{y[+mDint2] w[BR.E.BR]=SUPor-P}JIL-1[Scim] ry[506]} were introgressed into the genetic background y[1]; bw[1]; e[4]; ci[1] ey[R] to generate the strains Mod(mdg4)/+, Su(var)205/+ and JIL-1/+. All females used in the introgression were collected within 7 hours after eclosion. For gene expression analyses, flies were grown in incubators at 25°C, 65% of relative humidity, and constant light. Newly emerged male adults flies harboring the mutations and the Y chromosomes Yohio and Ycongo(Mod(mdg4)/+;Yohio and Mod(mdg4)/+;Ycongo, Su(var)205/+;Yohio and Su(var)205/+;Ycongo, and JIL-1/+;Yohio and JIL-1/+;Ycongo) were collected and aged for 2 days at the same rearing condition before they were flash-frozen in liquid nitrogen. Four replicas of each sample were collected and stored at -80°C. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). The genetic interaction of the Y chromosome and the mutation was studied by the following contrasts: 1) male flies Mod(mdg4)/+;Yohio versus male flies Mod(mdg4)/+;Ycongo; 2) male flies Su(var)205/+;Yohio versus male flies Su(var)205/+;Ycongo; 3) male flies JIL-1/+;Yohio versus male flies JIL-1/+;Ycongo). As reference we used the same genetic background without the mutations [+/+;Ycongo (background 2) and +/+;Yohio (background 2)]. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma.
2013-05-01 | GSE46313 | GEO
Project description:Fly strains were obtained from the Bloomington Stock Center. The chromosomes containing the gene deletion mod(mdg4)L3101 {y[1] w[1118]; P{w[+mC]=lacW}mod(mdg4)[L3101]/TM3, Ser[1]}, HP1 {In(1)w[m4h]; Su(var)205[5]/In(2L)Cy,In(2R)Cy, Cy[1]}, mod(mdg4)G16853 {w[1118]; P{w[+mC]=EP}mod(mdg4)[G16853]/TM6C, Sb[1]}, and Jil-1Scim {y[1]; P{y[+mDint2] w[BR.E.BR]=SUPor-P}JIL-1[Scim] ry[506]} were introgressed into the genetic background y[1]; bw[1]; e[4]; ci[1] ey[R] to generate the strains Mod(mdg4)/+, Su(var)205/+ and JIL-1/+. All females used in the introgression were collected within 7 hours after eclosion. For gene expression analyses, flies were grown in incubators at 25°C, 65% of relative humidity, and constant light. Newly emerged male adults flies harboring the mutations and the Y chromosomes Yohio and Ycongo(Mod(mdg4)/+;Yohio and Mod(mdg4)/+;Ycongo, Su(var)205/+;Yohio and Su(var)205/+;Ycongo, and JIL-1/+;Yohio and JIL-1/+;Ycongo) were collected and aged for 2 days at the same rearing condition before they were flash-frozen in liquid nitrogen. Four replicas of each sample were collected and stored at -80°C. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). The genetic interaction of the Y chromosome and the mutation was studied by the following contrasts: 1) male flies Mod(mdg4)/+;Yohio versus male flies Mod(mdg4)/+;Ycongo; 2) male flies Su(var)205/+;Yohio versus male flies Su(var)205/+;Ycongo; 3) male flies JIL-1/+;Yohio versus male flies JIL-1/+;Ycongo). As reference we used the same genetic background without the mutations [+/+;Ycongo (background 2) and +/+;Yohio (background 2)]. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma. Dye "swaps," loop design.
2013-05-01 | E-GEOD-46313 | biostudies-arrayexpress
Project description:modENCODE_submission_4094 This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The White Lab is aiming to map the association of many of the non-histone chromosomal proteins on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested chromatin-binding protein. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Y cn bw sp; Developmental Stage: L3; Genotype: y[1] oc[R3.2]; Gr22b[1] Gr22d[1] cn[1] CG33964[R4.2] bw[1] sp[1]; LysC[1] lab[R4.2] MstProx[1] GstD5[1] Rh6[1]; Sex: Unknown; EXPERIMENTAL FACTORS: Developmental Stage L3; Strain Y cn bw sp; Antibody KW4-mod(mdg4)-D2 (target is mod(mdg4))
2013-08-28 | E-GEOD-50348 | biostudies-arrayexpress
Project description:Transcriptomic analysis and identification of differentially expressed genes in control vs KD Drosophila melanogaster ovaries. We compared gene expression profiles in Drosophila melanogaster ovaries in which the Snr1 or the mod(mdg4) gene have been selectively knocked down by tissues specific shRNA expression.
2021-12-10 | GSE174263 | GEO
Project description:The asynchronous timing of replication of different chromosome domains is essential for eukaryotic genome stability, but the mechanisms establishing replication timing programs remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) domains in polytene chromosomes. SUUR negatively regulates DNA replication fork progression across IH; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. In vitro, Mod(Mdg4) stimulates the ATPase activity of SUUR, although neither SUUR nor SUMM4 can remodel nucleosomes. Mod(Mdg4)-67.2 and SUUR distribution patterns in vivo partially overlap, and Mod(Mdg4) is required for a normal spatiotemporal distribution of SUUR in chromosomes. SUUR and Mod(Mdg4)-67.2 mediate insulator activities of the gypsy mobile element that disrupt enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, mutations of SuUR or mod(mdg4) reverse the locus-specific UR. These findings reveal that DNA replication can be delayed by a chromatin barrier and thus, uncover a critical role for architectural proteins in replication timing control. They also provide a biochemical link between ATP-dependent motor factors and the activity of insulators in regulation of gene expression and chromatin partitioning.
2022-12-04 | GSE189421 | GEO