Negative regulation of Drosophila Transformer2 by SR splicing factors

The Drosophila Transformer-2 (Tra2) protein activates the splicing of doublesex and fruitless pre-mRNA and represses M1 intron splicing in its own RNA in male germline. The M1 retention is part of negative feedback mechanism that controls Tra2 protein synthesis. However it is not known how the M1 intron is repressed or why Tra2 activates splicing of some RNAs while repressing splicing in others. Here we show that Tra2 and SR protein Rbp1 function together to specifically repress M1 splicing in vitro through the same intronic silencer by binding independently to distinct sites. The role of Rbp1 in M1 repression in vivo was validated by the finding that increased expression of Rbp1 in S2 cells promotes M1 retention. Furthermore, Tra2 blocks prespliceosomal A complex formation, a step corresponding to U2 snRNP recruitment to the branchpoint. High levels of Tra2 repression require an upstream enhancer. Together, we propose that the complex formed by Tra2 and Rbp1 on the silencer achieves splicing repression by blocking the recognition of the branchpoint or antagonizing enhancer function. ^ In addition, both splicing regulatory activities of Tra2 are essential developmental events, doublesex splicing is the key for Drosophila sex determination in the soma, while M1 retention occurs in the male germline and is necessary for spermatogenesis. However, active Tra2 is expressed ubiquitously. So another issue we have studied is how Tra2 accomplishes negative and positive splicing regulation in a tissue-specific fashion. Surprisingly, we found that nuclear extract from somatically-derived S2 cells support M1 repression in vitro. This led us to hypothesize that no germline specific factor is required and that high levels of Tra2 expression in the male germline is sufficient to trigger M1 retention. To test it, I examined whether increased expression of Tra2 could promote M1 retention in cells outside male germline. My results show that increased Tra2 expression promotes M1 retention in somatically-derived S2 cells as well as in the somatic tissues of living flies. These results show that somatic tissues are capable of supporting M1 repression but do not normally do so because the low levels of Tra2 do not trigger negative feedback regulation. ^

Dysregulation of alternative splicing of coagulation factor V results in bleeding disorder, east Texas type and other disorders of hemostasis

The studies completed herein explore different phenotypes related to the genetic defects that predispose individuals to a disruption of normal hemostasis. In the first study, a novel autosomal dominant bleeding disorder, which is characterized by excessive bleeding with trauma or surgery and menorrhagia in affected women, was studied in a large family (16 affected individuals) from east Texas. Affected members had a prolongation of their PT and/or aPTT, but normal clinical coagulation studies. Previous linkage analysis by Kuang et. al. (2001) mapped the defective gene to 1g23-24 (LODmax 7.22), which contains the gene for coagulation factor V (FV). I identified an alteration (A2440G) in the FV gene in exon 13 that segregated with the disease and was not present in 62 controls. Interestingly, this alteration resulted in a 22-fold up-regulation of a novel alternative splicing variant in patients' RNA versus controls. This translated into a similar fold increase in a 250-kDa isoform of FV seen in patients' plasma versus controls. A recombinant of this splicing event exhibited an increased sensitivity to cleavage by activated protein C (APC) that was more striking in the presence of PS. In addition, this novel isoform had increased APC cofactor activity, thus increasing the degradation of FVIIIa. These data indicated that A2440G up-regulates an alternatively spliced transcript of FV, and increases a FV isoform that hinders coagulation as opposed to promoting it like its wild-type counterpart. ^ The second study reports the largest screening to date of African Americans in two independent cohorts for a rare prothrombin variant, C20209T, which is suspected to be associated with thrombotic disease. The Texas Medical Center Genetics Resource (TexGen) Stroke DNA repository revealed 1.67% (Fisher p=0.27) of African American stroke patients were heterozygous for the 20209*T allele. Screening of the Atherosclerosis Risk in Communities Study (ARIC) cohort (n=3470) for the 20209*T allele revealed a population prevalence of 0.58% in individuals of African American descent; however, all associations with thrombotic disease were negative. Analysis of these two independent cohorts revealed that, unlike its neighbor G20210A, the C20209T variant does not increase the risk of thrombotic events in the African American population. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Regulation of mRNA splicing in Moloney murine sarcoma virus mutants

Cells infected with the conditionally defective MuSVts110 mutant of Moloney murine sarcoma virus are transformed at 33$\sp\circ$C but appear morphologically normal at 39$\sp\circ$C. The molecular basis for this phenotype is as follows: MuSVts110 contains a 1487 nucleotide central deletion that has truncated the 3$\sp\prime$ end to the gag gene and the 5$\sp\prime$ end of the mos gene. The resulting gag-mos junction is out-of-frame and the v-mos protein is not expressed. At 33$\sp\circ$C or lower, a splicing event is activated such that a 431 base intron is removed to realign the gag and mos gene in-frame, allowing the expression of a transforming protein P85$\sp{gag-mos}$. Temperature-dependent splicing appeared to be an intrinsic property of MuSVts110 transcripts and not a general feature of pre-mRNA splicing in 6m2 cells since splicing activity of a heterologous transcript in the same cells did not vary with temperature. The possibility that the splice event was not temperature-sensitive, but that the accumulation of spliced transcript at the lower growth temperatures was due to its selective thermolability was ruled out as stability studies revealed that the relative turnover rates of the unspliced and spliced MuSVts110 transcripts were not affected by temperature.^ The consensus sequences containing the splice sites activated in the MuSVts110 mutant (5$\sp\prime$ gag and 3$\sp\prime$ mos) are present, but not utilized, in wild-type MuSV-124. To test the hypothesis that it was the reduction of the 1919 base intervening sequence in MuSV-124 to 431 bases in MuSVts110 which activated splicing, the identical 1487 base deletion was introduced into cloned wild-type MuSV-124 DNA to create the MuSVts110 equivalent, ts32.^ To examine conditions permissive for splicing, we assayed splice site activation in a series of MuSV-124 "intron-modification" mutants. Data suggest that splicing in wild-type MuSV-124 may be blocked due to the lack of a proximal branchpoint sequence, but can be activated by those intron mutations which reposition a branch site closer to the 3$\sp\prime$ splice site. (Abstract shortened with permission of author.) ^

{\it cis\/} -acting determinants in the control of MuSVts110 RNA splicing

Like other simple retroviruses the murine sarcoma virus ts110 (MuSVts110) displays an inefficient mode of genome splicing. But, unlike the splicing phenotypic of other retroviruses, the splicing event effected upon the transcript of MuSVts110 is temperature sensitive. Previous work in this laboratory has established that the conditionally defective nature of MuSVts110 RNA splicing is mediated in cis by features in the viral transcript. Here we show that the 5$\sp\prime$ splice site of the MuSVts110 transcript acts as a point of control of the overall splicing efficiency at both permissive and nonpermissive temperatures for splicing. We strengthened and simultaneously weakened the nucleotide structure of the 5$\sp\prime$ splice site in an attempt to elucidate the differential effects each of the two known critical splicing components which interact with the 5$\sp\prime$ splice site have on the overall efficiency of intron excision. We found that a transversion of the sixth nucleotide, resulting in the formation of a near-consensus 5$\sp\prime$ splice site, dramatically increased the overall efficiency of MuSVts110 RNA splicing and abrogated the thermosensitive nature of this splicing event. Various secondary mutations within this original transversion mutant, designed to selectively decrease specific splicing component interactions, lead to recovery of inefficient and thermosensitive splicing. We have further shown that a sequence of 415 nucleotides lying in the downstream exon of the viral RNA and hypothesized to act as an element in the temperature-dependent inhibition of splicing displays a functional redundancy throughout its length; loss and/or replacement of any one sequence of 100 nucleotides within this sequence does not, with one exception detailed below, diminish the degree to which MuSVts110 RNA is inhibited to splice at the restrictive temperature. One specific deletion, though, fortuitously juxtaposed and activated cryptic consensus splicing signals for the excision of a cryptic intron within the downstream exon and markedly potentiated--across a newly defined cryptic exon--the splicing event effected upon the upstream, native intron. We have exploited this mutant of MuSVts110 to further an understanding of the process of exon definition and intron definition and show that the polypyrimidine tract and consensus 3$\sp\prime$ splice site, as well as the 5$\sp\prime$ splice site, within the intron at the 3$\sp\prime$ flank of the defined exon are required for the exon's definition; implying that definition of the downstream intron is required for the in vivo definition of the proximal, upstream exon. Finally; we have shown, through the construction of heterologous mutants of MuSVts110 employing a foreign 3$\sp\prime$ end-forming sequence, that efficiency of transcript splicing can be increased--to a degree which abrogates its thermosensitive nature--in direct proportion to increasing proximity of the 3$\sp\prime$ end-forming signal to the terminal 3$\sp\prime$ splice site. ^

Thermo -modulation of MuSVts110 splicing by inhibitory RNA sequences

Viral systems have contributed tremendously to the understanding of eukaryotic molecular biology. The proportional pattern of retroviral RNA expression offers many clues into the alternative splicing of cellular transcripts. The MuSVts110 virus presents an unusual expression system, where the mechanistic combination of RNA splicing and cellular transformation can be physiologically manipulated. Splicing of MuSVts110 pre-mRNA occurs inefficiently (30%-50%) at 33$\sp\circ$C or below and is subdued at 39$\sp\circ$C ($<$5%). Like most alternatively spliced cellular and retroviral transcripts, the MuSVts110 pre-mRNA contains cis-acting intron and exon sequences that attenuate splicing. These include a splicing inhibitory sequence at the 3$\prime$ end of the MuSVts110 v-mos exon, called the E2 Distal Element (E2DE), and a sub-optimal 3$\prime$ splice site. The E2DE directly inhibits MuSVts110 RNA splicing in a sequence-specific fashion at 39$\sp\circ$C but not at 28$\sp\circ$C, potentially through the association of cellular factors. Inefficient MuSVts110 splicing is pre-dominantly attributed to the utilization of multiple weak branchpoint sequences located between $-113$ and $-34$ nucleotides upstream of the 3$\prime$ splice site. The molecular control of MuSVts110 splicing, represented primarily by scattered multiple inefficient branchpoint sequences that are conditionally modulated by the E2DE at higher growth temperatures, is discussed. ^

Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^

Accepting splicing systems with permitting and forbidding words

Abstract: In this paper we propose a generalization of the accepting splicingsystems introduced in Mitrana et al. (Theor Comput Sci 411:2414?2422,2010). More precisely, the input word is accepted as soon as a permittingword is obtained provided that no forbidding word has been obtained sofar, otherwise it is rejected. Note that in the new variant of acceptingsplicing system the input word is rejected if either no permitting word isever generated (like in Mitrana et al. in Theor Comput Sci 411:2414?2422,2010) or a forbidding word has been generated and no permitting wordhad been generated before. We investigate the computational power ofthe new variants of accepting splicing systems and the interrelationshipsamong them. We show that the new condition strictly increases thecomputational power of accepting splicing systems. Although there areregular languages that cannot be accepted by any of the splicing systemsconsidered here, the new variants can accept non-regular and even non-context-free languages, a situation that is not very common in the case of(extended) finite splicing systems without additional restrictions. We alsoshow that the smallest class of languages out of the four classes definedby accepting splicing systems is strictly included in the class of context-free languages. Solutions to a few decidability problems are immediatelyderived from the proof of this result.

Splicing Systems: Accepting Versus Generating.

In this paper we propose a condition for rejecting the input word by an accepting splicing system which is defined by a finite set of forbidding words. We investigate the computational power of the new variants of accepting splicing systems. We show that the new condition strictly increases the computational power of accepting splicing systems. Rather surprisingly, accepting splicing systems considered here can accept non-regular languages, a situation that has never occurred in the case of (extended) finite splicing systems without additional restrictions.

Rule Representation in Distributed Environments with Accepting Networks of Splicing Processors.

This paper presents the model named Accepting Networks of Evolutionary Processors as NP-problem solver inspired in the biological DNA operations. A processor has a rules set, splicing rules in this model,an object multiset and a filters set. Rules can be applied in parallel since there exists a large number of copies of objects in the multiset. Processors can form a graph in order to solve a given problem. This paper shows the network configuration in order to solve the SAT problem using linear resources and time. A rule representation arquitecture in distributed environments can be easily implemented using these networks of processors, such as decision support systems, as shown in the paper.

Aberrant intracellular localization of Varicella-Zoster virus regulatory proteins during latency

Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus.

Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver

Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215–222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5′ or the 3′ adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected Mr are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.

A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease

Some group I introns self-splice in vitro, but almost all are thought to be assisted by proteins in vivo. Mutational analysis has shown that the splicing of certain group I introns depends upon a maturase protein encoded by the intron itself. However the effect of a protein on splicing can be indirect. We now provide evidence that a mitochondrial intron-encoded protein from Aspergillus nidulans directly facilitates splicing in vitro. This demonstrates that a maturase is an RNA splicing protein. The protein-assisted reaction is as fast as that of any other known group I intron. Interestingly the protein is also a DNA endonuclease, an activity required for intron mobilization. Mobile elements frequently encode proteins that promote their propagation. Intron-encoded proteins that also assist RNA splicing would facilitate both the transposition and horizontal transmission of introns.

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.