Alanine repeats influence protein localization in splicing speckles and paraspeckles

Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated—fully or partially—from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.     

Solution Structure of the Second RRM Domain of RBM5 and Its Unusual Binding Characters for Different RNA Targets

The RNA binding motif protein 5 (RBM5), also known as LUCA15 or H37, containing two RNA recognition motifs, is a component of the spliceosome A complex. Previously, it has been reported that RBM5 bound to a U/C-rich sequence upstream of the In100 element at intron 9 of caspase2 pre-mRNA that enhanced the formation of proapoptotic caspase-2L isoform. In the present study, we solved the solution structure of the RBM5 RRM2 core domain and characterized its unusual binding capability for different RNA sequences. We found that the RBM5 RRM2 could preferentially bind to both CU rich and GA rich sequences with affinity in 10(-5) molar range. Further NMR experiments revealed that the dual RNA molecules could be accommodated on almost the same region of the protein's β-sheet surface and that both the N- and C-terminal regions of the protein were involved in the recognition. Our studies provide evidence for the RBM5 sequence specific interaction with the cis-acting element in pre-mRNA regulating alternative splicing.     

A novel splicing regulator shares a nuclear import pathway with SR proteins

Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin beta-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.     

Exon selection in alpha-tropomyosin mRNA is regulated by the antagonistic action of RBM4 and PTB

RNA-binding motif protein 4 (RBM4) has been implicated in the regulation of precursor mRNA splicing. Using differential display analysis, we identified mRNAs that associate with RBM4-containing messenger RNPs in vivo. Among these mRNAs, alpha-tropomyosin (alpha-TM) is known to exhibit a muscle cell type-specific splicing pattern. The level of the skeletal muscle-specific alpha-TM mRNA isoform partially correlated with that of RBM4 in human tissues examined and could be modulated by ectopic overexpression or suppression of RBM4. These results indicated that RBM4 directly influences the expression of the skeletal muscle-specific alpha-TM isoform. Using minigenes, we demonstrated that RBM4 can activate the selection of skeletal muscle-specific exons, possibly via binding to intronic pyrimidine-rich elements. By contrast, the splicing regulator polypyrimidine tract binding protein (PTB) excluded these exons; moreover, RBM4 antagonized this PTB-mediated exon exclusion likely by competing with PTB for binding to a CU-rich element. This study suggests a possible mechanism underlying the regulated alternative splicing of alpha-TM by the antagonistic splicing regulators RBM4 and PTB.     

Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors

The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF⁶⁵. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF⁶⁵ and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF⁶⁵, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF⁶⁵ in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.     

RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion

Tau protein, which binds to and stabilizes microtubules, is critical for neuronal survival and function. In the human brain, tau pre-mRNA splicing is regulated to maintain a delicate balance of exon 10-containing and exon 10-skipping isoforms. Splicing mutations affecting tau exon 10 alternative splicing lead to tauopathies, a group of neurodegenerative disorders including dementia. Molecular mechanisms regulating tau alternative splicing remain to be elucidated. In this study, we have developed an expression cloning strategy to identify splicing factors that stimulate tau exon 10 inclusion. Using this expression cloning approach, we have identified a previously unknown tau exon 10 splicing regulator, RBM4 (RNA binding motif protein 4). In cells transfected with a tau minigene, RBM4 overexpression leads to an increased inclusion of exon 10, whereas RBM4 down-regulation decreases exon 10 inclusion. The activity of RBM4 in stimulating tau exon 10 inclusion is abolished by mutations in its RNA-binding domain. A putative intronic splicing enhancer located in intron 10 of the tau gene is required for the splicing stimulatory activity of RBM4. Immunohistological analyses reveal that RBM4 is expressed in the human brain regions affected in tauopathy, including the hippocampus and frontal cortex. Our study demonstrates that RBM4 is involved in tau exon 10 alternative splicing. Our work also suggests that down-regulating tau exon 10 splicing activators, such as RBM4, may be of therapeutic potential in tauopathies involving excessive tau exon 10 inclusion.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

水稻NBS-LRR基因选择性剪接的全基因组检测及分析

选择性剪接是促进基因组复杂性和蛋白质组多样性的一种主要机制,但是对水稻NBS-LRR序列选择性剪接的全基因组分析却未见报道。通过隐马尔柯夫模型搜索,从TIGR数据库里得到了855条编码NBS-LRR基序的序列。利用这些序列在KOME、TIGR基因索引及UniProt三个数据库中进行同源搜索,获得同源的完整cDNA序列、假设一致性序列和蛋白质序列。再利用Spidey和SIM4程序把完整cDNA序列和假设一致性序列联配到相应的BAC序列上来预测选择性剪接。蛋白质序列和基因组序列之间的联配使用tBLASTn。在这875个NBS-LRR基因中,119个基因具有选择性剪接现象,其中包括71内含子保留,20个外显子跳跃,25个选择性起始,16个选择性终止,12个5′端的选择性剪接和16个3′端选择性剪接。大多数选择性剪接都为两个和多个转录本所支持。可以通过访问http://www.bioinfor.org查询这些数据。进而通过生物信息学分析剪接边界发现外显子跳跃和内含子保留的‘GT…AG’的规则不如组成型的保守。这暗示了它们是通过不同的调控机制来指导剪接变构体的形成。通过分析内含子保留对蛋白质的影响,发现选择性剪接的蛋白更倾向于改变其C端氨基酸序列。最后对选择性剪接的组织分布和蛋白质定位进行分析,结果表明选择性剪接的最大类的组织分布是根和愈伤组织。超过1/3剪接变构体的蛋白质定位是质膜和细胞质。这些选择性剪接蛋白可能在抗病信号转导中起到重要作用。     

Differential isoform expression and interaction with the P32 regulatory protein controls the subcellular localization of the splicing factor U2AF26

The U2 auxiliary factor (U2AF) is an integral part of the spliceosome that is important for the recognition of the 3' splice site. U2AF consists of a large and a small subunit, the prototypes of which are U2AF65 and U2AF35. Recent evidence suggests that several homologs of both U2AF subunits exist that are able to regulate alternative splicing. Here we have investigated the expression, intracellular localization, and nucleo-cytoplasmic shuttling of one homolog of the small U2AF subunit, U2AF26, and a splice variant lacking exon 7, U2AF26DeltaE7. In contrast to the nuclear U2AF26, which displays active nucleo-cytoplasmic shuttling, U2AF26DeltaE7 is localized in the cytoplasm. Our studies reveal a nuclear localization sequence in the C-terminal exons 7 and 8 of U2AF26 that differs from the known nuclear localization sequence in U2AF35. In addition, we could identify P32 as a protein that is able to interact with U2AF26 through this domain, and we demonstrate that this interaction is required for the nuclear translocation of U2AF26. Our results suggest the existence of two distinct nuclear import pathways for U2AF26 and U2AF35 that could independently control their intracellular distribution and availability to the splicing machinery. Such a mechanism could work in addition to the differential expression of U2AF homologs to contribute to the regulation of alternative splicing.     

Regulation of NF-kappa B2 p100 processing by its cis-acting domain

Processing of NF-kappa B2 precursor protein p100 to generate p52 is tightly regulated. However, this proteolytic event could be actively induced by the NF-kappa B-inducing kinase and the human T-cell leukemia virus-encoded oncoprotein Tax or be constitutively turned on due to the loss of the C-terminal portion of p100. Whereas NF-kappa B-inducing kinase-mediated p100 processing requires beta-transducin repeat-containing protein, constitutive processing of p100 is independent of this protein. On the other hand, Tax-induced processing of p100 appears to be both beta-transducin repeat-containing protein-dependent and -independent. We show here that, besides the C-terminal sequences, multiple functional regions, including the two alpha-helices, dimerization domain, nuclear localization sequence, and glycine-rich region, located in the N terminus of p100, also play important roles in both constitutive and inducible processing, suggesting a common mechanism for p100 processing. We further demonstrate that with the help of the C-terminal death domain and I kappa B kinase alpha-targeting serines, the C-terminal ankyrin-repeat domain of p100 strongly interacts with its N-terminal dimerization domain and nuclear localization sequence, thereby bringing the C- and N-terminal sequences together to form a three-dimensional domain. This presumptive domain is not only responsible for suppression of constitutive processing but also required for inducible processing of p100. Taken together, these studies highlight the mechanism by which the different sequences within p100 work in concert to regulate its processing and shed light on the mechanisms of how p100 processing is tightly and delicately controlled.     

Shuttling of SOX proteins

The control of access of SOX proteins to their nuclear target genes is a powerful strategy to activate or repress complex genetic programs. The sub-cellular targeting sequences of SOX proteins are concentrated within the DNA binding motif, the HMG (for high mobility group) domain. Each SOX protein displays two different nuclear localization signals located at the N-terminal and C-terminal part of their highly conserved DNA binding domain. The N-terminal nuclear localization signal binds calmodulin and is potentially regulated by intracellular calcium signalling, while the C-terminal nuclear localization signal, which binds importin-β, responds to other signalling pathways such as cyclic AMP/protein kinase A. Mutations inducing developmental disorders like sex reversal have been reported in both NLSs of SRY, interfering with its nuclear localization and suggesting that both functional nuclear localization signal are required for its nuclear activity. A nuclear export signal is also present in the HMG box of SOX proteins. Group E SOX proteins harbour a perfect consensus nuclear export signal sequence in contrast to all other SOX proteins, which display only imperfect ones. However, observations made during mouse embryonic development suggest that non-group E SOX proteins could also be regulated by a nuclear export mechanism. The presence of nuclear localization and nuclear export signal sequences confers nucleocytoplasmic shuttling properties to SOX proteins, and suggests that cellular events regulated by SOX proteins are highly dynamic.     

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.     

Abscisic acid does not influence the subcellular distribution of the HYL1 protein from Arabidopsis thaliana

HYL1 is a nuclear protein involved in the processing of miRNAs but its exact function remains unknown. Arabidopsis thaliana hyl1 mutants exhibit hypersensitivity to ABA. We decided to answer the question whether ABA affects the HYL1 protein localization within the cell and show that it does not. We also studied the expression of HYL1 in different tissues and organs. In this paper we show for the first time the expression profile of the HYL1 protein using anti-HYL1 antibodies. The protein is present in seedlings and mature plants in all organs studied, with the highest amount in inflorescences. A. thaliana HYL1 protein has several repetitions of a 28-amino-acid sequence at the C-terminus that confer protein instability. Our bioinformatic analysis of HYL1 homologs in different Brassica species shows that this repetition is typical only for Arabidopsis. This may suggest a relatively late evolutionary acquisition of the C-terminal domain.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C backbone and side chain resonance assignments of the RRM domain from human RBM24

Tissue development requires the expression of a regulated subset of genes, and it is becoming clear that the process of alternative splicing also plays an important role in the production of necessary tissue-specific isoforms. However, only a few of these tissue-specific splicing factors in mammals have so far been discovered. One of these factors is the RNA-binding protein RBM24 which has been recently identified as a major regulator of alternative splicing in cardiac and skeletal muscle development. The RBM24 protein contains an RNA recognition motif (RRM) domain that presumably mediates the binding to target pre-mRNA required for regulation of the splicing patterns. Here we report ¹H, ¹⁵N and ¹³C chemical shift assignments of the backbone and sidechain atoms for the RRM domain from human RBM24. Secondary chemical shift analysis and relaxation measurement confirm the canonical architecture of the RRM domain. The data will allow for atomic level studies aimed at understanding splicing regulation of target genes in heart and muscle development and investigation into a separate role of RBM24 in modulating mRNA stability of genes involved in the p53 tumor suppressor pathway.