transformer 2


EVOLUTIONARY HOMOLOGS (part 1/3)

SR proteins are RNA binding proteins with a conserved serine/arginine domain involved in protein-protein interactions. Included among SR proteins are a group of highly conserved proteins, numbering six or more, involved in the alternative splicing of many pre-messenger RNAs. In addition to what appears to be a highly conserved repertoire of SR proteins, a number or SR proteins with very specialized functions have been identified. These include TRA and TRA2 of Drosophila, involved in the sex determination hierarchy. The literature below represents a sample of the SR protein literature, and includes some information on the highly conserved proteins, information on proteins that interact with SR proteins, and several examples of SR proteins with specialized functions.

Tra2 in other Drosophila species and other SR proteins in insects

The splicing factor TRA-2 affects sex-specific splicing of multiple pre-mRNAs involved in sexual differentiation. The tra-2 gene itself expresses a complex set of mRNAs generated through alternative processing that collectively encode three distinct protein isoforms. The expression of these isoforms differs in the soma and germ line. In the male germ line the ratio of the two isoforms present is governed by autoregulation of splicing. However, the functional significance of multiple TRA-2 isoforms has remained uncertain. The structure, function, and regulation of tra-2 were examined inDrosophila virilis (DV), a species diverged from D. melanogaster by over 60 million years. The DV homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in DM. When introduced into the genome of DM, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative mRNAs produced in DV testes suggests that germ line-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in DM. The similarity in structure and function of the tra-2 genes in these divergent Drosophila species supports the idea that sexual differentiation in DM and DV is accomplished under the control of similar regulatory pathways (Chandler, 1997).

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Dominant and loss-of-function alleles were isolated for B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at Doublesex mRNA under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. A comparison of B52ED with the B52+ allele from which it was derived reveals a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele using X rays leads to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo (Peng, 1995).

Balbiani rings, the most active genes in the polytene chromosomes of the midge Chironomus (Diptera) code for secretory giant peptides. A heterogeneous nuclear ribonucleoprotein (hnRNP), Ct-hrp45, is one of the major components of pre-mRNP particles in Chironomus tentans. hrp45 belongs to the SR family of splicing factors and exhibits high sequence similarity to Drosophila SRp55/B52 and human SF2/ASF. The distribution of hrp45 within the C. tentans salivary gland cells has been found to be abundant in the nucleus, whereas it is undetectable in the cytoplasm. The fate of hrp45 in specific pre-mRNP particles (the Balbiani ring (BR) granules), has been revealed by immunoelectron microscopy. hrp45 is associated with the growing BR pre-mRNP particles and is added continuously concomitant with the growth of the transcript, indicating that hrp45 is bound extensively to exon 4, which comprises 80-90% of the primary transcript. Furthermore, hrp45 remains bound to the BR RNP particles in the nucleoplasm and is not released until the particles translocate through the nuclear pore. Thus, hrp45 behaves as an hnRNP protein linked to exon RNA (and perhaps also to the introns) rather than as a spliceosome component connected to the assembly and disassembly of spliceosomes. It seems that hrp45, and possibly also other SR family proteins, is playing an important role in the structural organization of pre-mRNP particles and is perhaps participating not only in splicing but also in other intranuclear events (Alzhanova-Ericsson, 1996).

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). The Ceratitis homolog of Sxl does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent, which is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, dsx, is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation. The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx revealed the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to TRA and TRA-2. Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. It is proposed that this repression is achieved by M, the Y-linked male-determining factor (Pane, 2002).

The results show that Ceratitis and Drosophila sex-determining cascades share a conserved tra-->dsx genetic module to control sex determination and sexual differentiation as well as that tra sex-specific splicing regulation differs in the two species. In Drosophila, TRA protein, together with TRA-2, binds to the TRA/TRA-2 recognition sequences on the Drosophila dsx pre-mRNA and promotes the use of a nearby female-specific acceptor site. Cctra is needed to impose the female-specific splicing of Ccdsx, most probably by a similar mechanism as in Drosophila, invoking the existence of a Cctra2 homolog. This hypothesis is also supported by the finding of TRA/TRA-2 recognition sequences located in close vicinity to the female-specific acceptor site in Ccdsx pre-mRNA (Pane, 2002).

In Drosophila, tra female-specific splicing is promoted by SXL, which blocks the use of the non-sex-specific splice site present in the tra pre-mRNA. In Ceratitis, the presence of multiple TRA/TRA-2-binding elements within the Cctra male-specific exonic sequences strongly suggests that CcTRA and a hypothetical CcTRA-2 protein could bind to these sequences, thus mediating a direct autoregulation. The unusually strong phenotypic effects of the RNAi against this gene also support this model of Cctra regulation. The localization of the putative regulatory elements within the Cctra gene indicates a repression mode by which CcTRA in females prevents the recognition of male-specific splice sites. The mechanism by which Cctra seems to promote the female mode of processing of its own pre-mRNA by TRA/TRA-2-binding elements appears to be different also from the female-specific splicing of dsx. Rather than activating a splice site nearby the regulated exon, as in the case of dsx, inclusion of male-specific Cctra sequences is suppressed when CcTRA is present. Although this would be a novelty with respect to known Drosophila TRA/TRA-2 activities, it has been previously shown that the 'behavior' of these cis elements is context dependent and that changing the location of splicing enhancers can transform them into negative regulatory elements (Pane, 2002).

In Drosophila, the presence of the Y chromosome is necessary for male fertility but not for male development. By contrast, RNAi-treated Ceratitis embryos with a female XX karyotype can develop into fertile males, which indicates that transient repression of Cctra by RNAi is sufficient to implement fully normal male development. The cases of complete sexual transformation of genetic Ceratitis females (XX) into fertile males by RNAi demonstrate that the Y chromosome, except for the dominant male determiner M, does not supply any other contribution to both somatic and germline male development, as suggested by previous Y-chromosome deletion analysis. Other dipteran species, such as Musca domestica and Chrysomya rufifacies show a female and male germline sex determination that is completely dependent on the sexual fate of the soma. However, in Drosophila, the XX and XY germ cells seem to respond differently to sex determining somatic cues. Indeed the XY germ cells have also an autonomous stage-specific sex determination mechanism that probably integrates the somatic signal. In Ceratitis, Cctra could be required in XX somatic cells to let them induce the XX germ cells to differentiate as oogenic cells. Alternatively, Cctra could be required in XX germ cells to 'feminize' them. This case would be a novelty with respect to the known Drosophila transformer gene functions (Pane, 2002).

Since zygotes that carry a Y chromosome do not activate Cctra female-specific splicing and autoregulation, it is proposed that the Y-linked male-determining M factor prevents this activation. It is conceivable that Cctra is a direct target of the M factor. Presence of this M factor in the zygote may prevent the production of CcTRA protein. The Cctra positive feedback loop is a probable target for regulation, because of its sensitivity (already shown by RNAi). An important question to be addressed is how autoregulation of Cctra is initiated in XX embryos of C. capitata and how this is prevented in XY embryos. A possible explanation is suggested by the Cctra female-specific mRNAs encoding the full-length protein, which have been detected in unfertilized eggs. Depositing these Cctra transcripts in eggs may provide a source of activity that can be used later for 'female-specific' processing when Cctra is zygotically transcribed. Once zygotically activated in XX embryos, Cctra promotes its own female-specific splicing, maintaining the female sex determination and the female-specific splicing of the downstream Ccdsx gene. Taken together, these events induce the female differentiation. In the current model for sex determination of medfly, the M factor is directly involved in the Cctra sex-specific regulation. Thus, in the presence of M Cctra, autoregulation is blocked and the gene produces male-specific transcripts encoding short and possibly non-functional CcTRA peptides. The absence of CcTRA leads Ccdsx to produce male-specific transcripts by default, promoting male differentiation. The control of the M factor upon Cctra expression could be exerted at different levels. The male determiner M could, for example, act at the pre-translational level blocking the production of CcTRA protein from the maternal transcripts. M could act at the post-translational level antagonizing the formation of protein complexes necessary for the female splicing mode. Or M could act as a transient transcriptional repressor of Cctra to reduce the amount of active CcTRA below a threshold needed to maintain the feedback loop. The proposed autoregulatory model of Cctra may also explain the remarkable efficiency of sex reversal by Cctra RNAi: a transient silencing of Cctra by injecting dsRNA is sufficient to let the loop collapse. Furthermore, the sensitivity of this positive autoregulation could be an evolutionary widely conserved pre-requisite to permit a 'faster' recruitment/replacement of different upstream regulators and to easily evolve different sex determining primary signals, as observed in dipteran species (Pane, 2002).

Sex can even be determined by a maternal effect in dipteran species such as Sciara coprophila and Chrysomya rufifacies. The hypothesis of a Cctra maternal contribution to the activation of the zygotic Cctra gene has similarities to the model of sex determination proposed for Musca domestica. In the common housefly, the maternal product of the key switch gene F is needed to activate the zygotic function of F in females. Musca male development results whenever F cannot become active in the zygote. This happens when the male-determining M is present in the zygotic genome, or when maternal F is not functional because of either the presence of M or the mutational loss of function of F (Fman) in the germline. More interesting, embryonic RNAi against the Musca tra-2 homolog causes sex reversion of Musca XX adults into intersex and fertile males, although this gene is not sex-specifically expressed. These recent data in Musca and the results in Ceratitis support the idea that F of Musca functionally corresponds to the Ceratitis tra gene, which seems to autoregulate and maternally contribute to its own activation, rather than to the Drosophila tra gene (Pane, 2002).

These data show that a basic structure of sex determination is conserved in the two dipteran species, namely the flow of 'instructions' from tra to dsx. This confirms the model of 'bottom-up' evolution, suggesting that during evolution developmental cascades are built from bottom up and that the genes at the bottom are widely conserved, while further upstream new regulatory elements may be recruited. The results show that Ceratitis and Drosophila sex-determining cascade differ at the level of transformer as well as upstream of it. Indeed the gene has conserved its function during evolution, but it has female-specific positive autoregulation in Ceratitis, while in Drosophila it needs Sxl as upstream regulator to express its female determining function. More likely the sex-determining function of Sxl was co-opted after Drosophila and Ceratitis had separated more than 100 Myr ago. Furthermore, it is conceivable that the autoregulatory mechanism of Sxl could have been selected to overcome a mutation impairing the tra autoregulation. Hence, in both species the female pathway is maintained by a single gene positive-feedback mechanism through sex-specific alternative splicing. Single gene autoregulation by alternative splicing seems not to be infrequent in nature, especially in those genes encoding splicing regulators. Indeed, other genes encoding RNA-binding proteins are thought to autoregulate their expression by controlling the processing of their own pre-mRNAs. Such a single-gene network with positive regulation is capable of bistability. This suggests that the emergence of analogous positive autoregulation in different genes such as Drosophila Sxl and Ceratitis tra genes would have been selected, during evolution, to guarantee a similar ON/OFF-female/male bistable cell state (Pane, 2002).

Since Ceratitis capitata is a major agricultural pest in many areas of the world, the isolation of a key sex-determining gene such as Cctra will substantially aid the development of new strategies to optimize the efficacy of currently used male sterile techniques for pest control. It is expected that tra is also a key sex-determining gene in many other insect species. Hence, the isolation of corresponding tra genes will open new means to control not only agricultural pests but also medically relevant vectors of diseases such as Glossina palpalis and Anopheles gambiae (Pane, 2002).

Mouse and Human Tra2 homologs

htra-2alpha is a human homolog of tra2. Two alternative types of htra-2alpha cDNA clones have been identified than encode different protein isoforms with striking organizational similarity to Drosophila tra2. Comparison of the D. melanogaster and D. virilis genes reveals an unrecognized but highly conserved region of 19 amino acids immediately downstream of the RRM, referred to as the linker region. The linker region is conserved in humans. When expressed in flies, one hTRA-2alpha isoform partially replaces the function of Drosophila TRA2, affecting both female sexual differentiation and alternative splicing of DSX pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2alpha to regulate dsx is female specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2alpha has conserved a striking degree of functional specificity during evolution and leads to the suggestion that the tra2 products of flies and humans have similar molecular functions, although they are likely to serve different roles in development (Dauwalder, 1996).

The SR protein B52 has been shown to be required for development in Drosophila; another SR protein, ASF/SF2, is required for viability of chicken DT40 cells, but Drosophila Tra2 is apparently nonessential. No functions for Tra2, outside sexual differentiation, have so far been discovered in Drosophila. Recently, two human homologs of Tra2, Tra2 and Tra2beta, have been identified. Using transgenic flies with nonfunctional Tra2, human Tra2 has been shown to be able to rescue Tra-dependent but not Tra-independent functions. The natural functions of mammalian Tra2 proteins are, however, unknown, as are their RNA binding specificities. Since the mechanisms of sexual differentiation are not conserved between mammals and flies, it is possible that human Tra2 proteins serve more general purposes than their Drosophila homolog (Tacke, 1998).

Human Tra2 proteins are present in HeLa cell nuclear extracts; they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. The purine rich ESE, AS3, consists of three copies of a high affinity ASF/SF2 binding site. Human Tra2 proteins bind specifically to A3. Indeed, the two purified Tra2 proteins bind preferentially to RNA sequences containing GAA repeats, which are characteristic of many enhancer elements. Neither Tra2 protein functions in constitutive splicing in vitro, and neither can substitute for the essential splicing function of SR proteins, but both activate enhancer-dependent splicing in a sequence-specific manner and restore it after inhibition with competitor RNA. These findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns (Tacke, 1998).

What are the specific functions of the two human Tra2 proteins in vivo? The data have not revealed functional differences between Tra2alpha and Tra2beta. Their RBDs are 85% identical, consistent with the finding that their RNA binding specificities are indistinguishable. The proteins differ mainly in the N-terminal 49 amino acids and in the position of a polyglycine stretch within their C-terminal RS domains, which otherwise are highly homologous. One possibility is that the two proteins are functionally redundant in vivo. Alternatively, the small differences in primary structure may account for potential differences in protein-protein interactions, including interaction with constitutive splicing factors such as SR proteins or with cell-specific factors yet to be discovered. An important aspect of the function of the Drosophila Tra protein in the regulation of dsx splicing appears to be its ability to influence the RNA binding properties of Tra2 through cooperative interaction. While Tra2 binds preferentially to the purine-rich element of the dsx enhancer in the absence of Tra, it can also bind to the dsx repeats in the presence of Tra. It is tempting to postulate a scenario in which cell-specific factors alter the RNA binding properties of human Tra2 proteins, analogous to the Tra/Tra2 cooperation in Drosophila. It is also noteworthy that Tra2beta was initially identified as a factor rapidly induced during reoxygenation of astrocytes after hypoxia and subsequently shown to display different mRNA expression levels in various mouse tissues. These examples raise the possibility that at least in some cases mammalian Tra2 proteins themselves might act as stage-specific regulators of pre-mRNA splicing. As in Drosophila, in vivo genetic studies may ultimately be required to elucidate the precise roles of mammalian Tra2 proteins in the control of cell- and stage-specific splicing patterns (Tacke, 1998).

Astrocytes have a critical role in the neuronal response to ischemia, sincetheir production of neurotrophic mediators can favorably impact on the extreme sensitivity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. RA301 is the same as Tra2beta, a sequence-specific activator of pre-mRNA splicing and a homolog of Drosophila Tra2. Analysis of the deduced amino acid sequence shows two ribonucleoprotein domains and serine/arginine-rich domains, suggestive of a function as an RNA splicing factor. Northern analysis displays striking induction only in cultured astrocytes within 15 min of reoxygenation and reaches a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrates expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/reoxygenated astrocytes. Induction of RA301 mRNA is mediated, in large part, by endogenously generated reactive oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase, which blocks oxygen-free radical formation by astrocytes. Similarly, increased expression of RA301 in supporting a neurotrophic function for astrocytes is suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expression is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues (Matsuo, 1995).

SIG41, a murine homolog of Drosophila TRA2, is a protein of 288 amino acids that is 45% identical to TRA2. There are four types of transcripts in mouse cells. Messenger RNA is present in virtually all cell lines and tissues studied, with remarkable levels in uterus and brain tissues. Differential stability of the SIG41 mRNAs is detected in mouse macrophage cells (Segade, 1996).

The role of SR proteins in splicing

Continued to transformer2: Evolutionary homologs part 2/3 | part 3/3


transformer 2: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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