7 research outputs found
Negative feedback regulation among SR splicing factors encoded by Rbp1 and Rbp1-like in Drosophila.
SR proteins constitute a widely conserved family of splicing regulators. Negative autoregulation of SR proteins has been proposed to exert homeostatic control on the splicing environment, but few examples have been studied and the role of isoforms that lack the RS domain is unclear. We show that genes Rbp1 and Rbp1-like, which encode Drosophila homologs of mammalian SRp20, negatively autoregulate and crossregulate at the level of alternative 3' splice site selection. This adjusts the relative expression of isoforms with either an RS domain or unrelated C-terminal domains (ALT) that are rich in serine and threonine. The effects of RBP1-ALT on splicing of doublesex and Rbp1-like are opposite to those of RBP1-RS and RBP1L-RS. RBP1-ALT and -RS exert opposing negative feedback on the ALT/RS ratio. However, RBP1-ALT inhibits the expression of RBP1-RS while stimulating that of RBP1L-RS. This asymmetry may contribute to changes in the RBP1-RS/RBP1L-RS ratio that are observed during development. These results provide the first example of a feedback-regulated SR protein network with evidence of an active homeostatic role for alternative isoforms.</p
Stabilization and analysis of intron lariats in vivo.
The analysis of lariats produced in vivo during pre-mRNA splicing is a powerful tool for elucidation of regulatory mechanisms and identification of natural recursive splicing events. Nevertheless, this analysis is technically challenging because lariats normally have short half-lives. With appropriate controls, RT-PCR amplification and sequencing of the region spanning the 2'-5' phosophodiester bond at the branch junction can be a sensitive and versatile method for lariat analysis. This approach can be facilitated and enhanced by reducing the activity of debranching enzyme (DBR) in order to stabilize lariats. We have generated a set of plasmids for dsRNA-mediated knockdown of DBR under diverse conditions in transgenic Drosophila and in cultured cells. We describe the use of these plasmids and protocols for lariat analysis. We have generated transgenic Drosophila strains carrying a GAL4-regulated RNAi construct that allows selective knockdown of DBR in specific tissues or developmental stages, using the large collection of available GAL4 expression lines. These strains should prove useful for detailed developmental analyses of alternative and recursive splicing and for genetic analyses of splicing factors. Similar approaches should be readily adaptable to other organisms.</p
Fluorescent PNA probes as hybridization labels for biological RNA.
Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts. Noncovalent strategies for labeling RNA rely on attaching fluorescent dyes to hybridization probes which deliver the dye to a specific region of the RNA through Watson-Crick base pairing. This report demonstrates the use of high-affinity peptide nucleic acid (PNA) probes in labeling mRNA transcripts with thiazole orange donor and Alexa-594 acceptor fluorophores. The PNA probes were targeted to sequences flanking splice sites in a pre-mRNA such that before splicing the PNAs were separated by >300 nucleotides (nts) whereas after splicing the separation decreased to</p
Identification of fat-cell enhancer regions in Drosophila melanogaster.
The insect fat body is a dynamic tissue involved in maintaining homeostasis. It functions not only in energy storage and intermediary metabolism but also in detoxification, communication and the immune response. Some of these functions are confined to distinct groups of fat body cells. In Drosophila melanogaster, discrete precursor-cell clusters populate the fat body [Hoshizaki, D.K., Blackburn, T., Price, C., Ghosh, M., Miles, K., Ragucci, M. and Sweis, R. (1994) Embryonic fat-cell lineage in Drosophila melanogaster. Development 120: 2489-2499; Hoshizaki, D.K., Lunz, R., Ghosh, M. and Johnson, W. (1995) Identification of fat-cell enhancer activity in Drosophila melanogaster using P-element enhancer traps. Genome 38: 497-506; Riechmann, V., Rehorn, K.P., Reuter, R. and Leptin, M. (1998) The genetic control of the distinction between fat body and gonadal mesoderm in Drosophila. Development 125: 713-723]. Whether these clusters populate defined morphological regions or whether they represent the precursors to functionally similar groups of fat-body cells has not been formally demonstrated. We have identified a 2.1 kb enhancer region from serpent (srp), a GATA transcription factor gene that is sufficient to induce fat-cell formation. This enhancer region drives expression in specific groups of precursor-cell clusters, which we show give rise to defined regions of the mature embryonic fat body. We present evidence that srp expression in different precursor fat cells is controlled by independent cis-acting regulatory regions, and we have tested the role of trans-acting factors in the specification of some of these cells. We suggest that the different positional cues regulating srp expression, and therefore general fat-cell specification, might also be involved in the functional specialization of fat cells. This may be a common mechanism in insects to explain the origin of biochemically distinct regions of the larval/adult fat body.</p
Subdivision of large introns in Drosophila by recursive splicing at nonexonic elements.
Many genes with important roles in development and disease contain exceptionally long introns, but special mechanisms for their expression have not been investigated. We present bioinformatic, phylogenetic, and experimental evidence in Drosophila for a mechanism that subdivides many large introns by recursive splicing at nonexonic elements and alternative exons. Recursive splice sites predicted with highly stringent criteria are found at much higher frequency than expected in the sense strands of introns >20 kb, but they are found only at the expected frequency on the antisense strands, and they are underrepresented within intronspseudoobscura, despite extensive divergence of other sequences within the same introns. These patterns of enrichment and conservation indicate that recursive splice sites are advantageous in the context of long introns. Experimental analyses of in vivo processing intermediates and lariat products from four large introns in the unrelated genes kuzbanian, outspread, and Ultrabithorax confirmed that these introns are removed by a series of recursive splicing steps using the predicted nonexonic sites. Mutation of nonexonic site RP3 within Ultrabithorax also confirmed that recursive splicing is the predominant processing pathway even with a shortened version of the intron. We discuss currently known and potential roles for recursive splicing.</p
Fine-mapping reveals novel alternative splicing of the dopamine transporter.
The dopamine transporter gene (SLC6A3, DAT) has been implicated in the pathogenesis of numerous psychiatric and neurodevelopmental disorders, including schizophrenia (SZ). We previously detected association between SZ and intronic SLC6A3 variants that replicated in two independent Caucasian samples, but had no obvious function. In follow-up analyses, we sequenced the coding and intronic regions of SLC6A3 to identify complete linkage disequilibrium patterns of common variations. We genotyped 78 polymorphisms, narrowing the potentially causal region to two correlated clusters of associated SNPs localized predominantly to introns 3 and 4. Our computational analysis of these intronic regions predicted a novel cassette exon within intron 3, designated E3b, which is conserved among primates. We confirmed alternative splicing of E3b in post-mortem human substantia nigra (SN). As E3b introduces multiple in-frame stop codons, the SLC6A3 open reading frame is truncated and the spliced product may undergo nonsense mediated decay. Thus, factors that increase E3b splicing could reduce the amount of unspliced product available for translation. Observations consistent with this prediction were made using cellular assays and in post-mortem human SN. In mini-gene constructs, the extent of splicing is also influenced by at least two common haplotypes, so the alternative splicing was evaluated in relation to SZ risk. Meta-analyses across genome-wide association studies did not support the initial associations and further post-mortem studies did not suggest case-control differences in splicing. These studies do not provide a compelling link to schizophrenia. However, the impact of the alternative splicing on other neuropsychiatric disorders should be investigated. © 2010 Wiley-Liss, Inc.</p
Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.
Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.</p