Sergiy Lopato

Proto-oncoprotein tls/fus is associated to the nuclear matrix and complexed with splicing factors ptb, srm160, and sr proteins

TLS/FUS is a nucleic acid-binding protein whose N-terminal half functions as a transcriptional activator domain in fusion oncoproteins found in human leukemias and liposarcomas. Previous reports have suggested a role for TLS/FUS in transcription and splicing processes. Here we report the association of TLS/FUS with the nuclear matrix and investigate its role in splicing. Splicing of two pre-mRNAs was inhibited in a TLS/FUS-immunodepleted extract and could only be partly restored by addition of recombinant TLS/FUS or/and SR proteins, known interaction partners of TLS/FUS. The subsequent analysis of TLS/FUS immunoprecipitates revealed that, in addition to the SR proteins SC35 and SRp75, the splicing factor PTB (hnRNPI) and the splicing coactivator SRm160 are complexed with TLS/FUS, thus explaining the inability to restore splicing completely. Coimmunolocalization confirmed the nuclear matrix association and interaction of TLS/FUS with PTB, SR proteins, and SRm160. Our results suggest that the matrix protein TLS/FUS plays a role in spliceosome assembly.

Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins

Alternative splicing is an important mechanism for fine tuning of gene expression at the post-transcriptional level. SR proteins govern splice site selection and spliceosome assembly. The Arabidopsis genome encodes 19 SR proteins, several of which have no orthologues in metazoan. Three of the plant specific subfamilies are characterized by the presence of a relatively long alternatively spliced intron located in their first RNA recognition motif, which potentially results in an extremely truncated protein. In atRSZ33, a member of the RS2Z subfamily, this alternative splicing event was shown to be autoregulated. Here we show that atRSp31, a member of the RS subfamily, does not autoregulate alternative splicing of its similarily positioned intron. Interestingly, this alternative splicing event is regulated by atRSZ33. We demonstrate that the positions of these long introns and their capability for alternative splicing are conserved from green algae to flowering plants. Moreover, in particular alternative splicing events the splicing signals are embedded into highly conserved sequences. In different taxa, these conserved sequences occur in at least one gene within a subfamily. The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions hidden in the genes of these plant-specific SR proteins.

Characterization of a novel arginine/serine-rich splicing factor in Arabidopsis.

Many splicing factors in vertebrate nuclei belong to a class of evolutionarily conserved proteins containing arginine/serine (RS) or serine/arginine (SR) domains. Previously, we demonstrated the existence of SR splicing factors in plants. In this article, we report on a novel member of this splicing factor family from Arabidopsis designated atRSp31. It has one N-terminal RNA recognition motif and a C-terminal RS domain highly enriched in arginines. The RNA recognition motif shows significant homology to all animal SR proteins identified to date, but the intermediate region does not show any homology to any other known protein. Subsequently, we characterized two cDNAs from Arabidopsis that are highly homologous to atRSp31 (designated atRSp35 and atRSp41). Their deduced amino acid sequences indicate that these proteins constitute a new family of RS domain splicing factors. Purified recombinant atRSp31 is able to restore splicing in SR protein-deficient human S100 extracts. This indicates that atRSp31 is a true plant splicing factor and plays a crucial role in splicing, similar to that of other RS splicing factors. All of the three genes are differentially expressed in a tissue-specific manner. The isolation of this new plant splicing factor family enlarges the essential group of RS domain splicing factors. Furthermore, because no animal equivalent to this protein family has been identified to date, our results suggest that these proteins play key roles in constitutive and alternative splicing in plants.

Network of Interactions of a Novel Plant-specific Arg/Ser-rich Protein, atRSZ33, with atSC35-like Splicing Factors

Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, anArabidopsis ortholog of human SF2/ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.