The role of alternative pre-mRNA splicing in regulating the structure and function of skeletal protein 4.1

Alternative pre-mRNA splicing plays a major role in regulating cell type-specific expression of the protein 4.1 family of skeletal proteins. The biological importance of alternative splicing as a mechanism for 4.1 gene regulation is underscored by studies of the prototypical 4.1R gene in erythroid cells: activation of exon 16 inclusion in mRna at the erythroblast stage greatly enhances the ability of newly synthesized 4.1R protein to bind spectrin and actin, and thus assemble into a stable membrane skeleton. This gain-of- function has profound effects on the biophysical properties of deformability and membrane strength that are critical to red cell survival in the circulation. Another example of developmentally regulated splicing occurs in differentiating mammary epithelial cells in culture, where cell morphogenesis is accompanied by a splicing switch that reversibly activates inclusion of alternative exon muscle. Few other genes are known to be so richly endowed with regulated switches in pre-mRna splicing making the 4.1R gene an interesting paradigm for the role of alternative splicing as a mediator of cell function. Recent evidence that other members of the 4.1 gene family are also regulated by alternative splicing suggests, moreover, that this phenomenon is of general importance in regulating the structure of this class of skeletal proteins.     

Members of the heterogeneous nuclear ribonucleoprotein H family activate splicing of an HIV-1 splicing substrate by promoting formation of ATP-dependent spliceosomal complexes

In this study we analyzed members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H protein family to determine their RNA binding specificities and roles in splicing regulation. Our data indicate that hnRNPs H, H', F, 2H9, and GRSF-1 bind the consensus motif DGGGD (where D is U, G, or A) and aggregate in a multimeric complex. We analyzed the role of these proteins in the splicing of a substrate derived from the HIV-1 tat gene and have shown that hnRNP H family members are required for efficient splicing of this substrate. The hnRNP H protein family members activated splicing of the viral substrate by promoting the formation of ATP-dependent spliceosomal complexes. Mutational analysis of six consensus motifs present within the intron of the substrate indicated that only one of these motifs acts as an intronic splicing enhancer.     

A novel family of plant splicing factors with a Zn knuckle motif: examination of RNA binding and splicing activities

An important group of splicing factors involved in constitutive and alternative splicing contain an arginine/serine (RS)-rich domain. We have previously demonstrated the existence of such factors in plants and report now on a new family of splicing factors (termed the RSZ family) from Arabidopsis thaliana which additionally harbor a Zn knuckle motif similar to the human splicing factor 9G8. Although only around 20 kDa in size, members of this family possess a multi-domain structure. In addition to the N-terminal RNA recognition motif (RRM), a Zn finger motif of the CCHC-type is inserted in an RGG-rich region; all three motifs are known to contribute to RNA binding. The C-terminal domain has a characteristic repeated structure which is very arginine-rich and centered around an SP dipeptide. One member of this family, atRSZp22, has been shown to be a phosphoprotein with properties similar to SR proteins. Furthermore, atRSZp22 was able to complement efficiently splicing deficient mammalian S100 as well as h9G8-depleted extracts. RNA binding assays to selected RNA sequences indicate an RNA binding specificity similar to the human splicing factors 9G8 and SRp20. Taken together, these result show that atRSZp22 is a true plant splicing factor which combines structural and functional features of both h9G8 and hSRp20.     

Axo-glial interactions regulate the localization of axonal paranodal proteins

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain- bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin beta/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.     

Alternative splicing generates variants in important functional domains of human slow skeletal troponin T

We provide the first nucleotide sequence information for the slow isoform of troponin T (TnT). Sequence and hybridization analyses revealed that a single slow TnT gene present in the human genome gives rise to at least two different slow TnT variants by alternative splicing. The observed variations in slow TnT splicing generated major structural differences between the two corresponding slow TnT proteins in a domain that is likely to be involved in critical interactions with troponin C, troponin I, and tropomyosin in the thin filament. Corresponding variations have not been found for fast or for cardiac TnT. The comparison of splicing patterns for fast, cardiac, and slow TnT reveals that the splicing pattern for each isoform is unique. These features raise important questions of why and how all the individual members of the closely related TnT gene family developed such complex but different schemes of alternative splicing to create sets of variant proteins. This unusual familial trait is not known in any other muscle or nonmuscle multigene family.     

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.     

Helicases involved in splicing from malaria parasite Plasmodium falciparum

An interesting element of eukaryotic genomes is the large quantity of non-coding intervening sequences commonly known as introns, which regularly interrupt functional genes and therefore must be removed prior to the use of genetic information by the cell. After splicing, the mature RNA is exported from the nucleus to the cytoplasm. Any error in the process of recognition and removal of introns, or splicing, would lead to change in genetic message and thus has potentially catastrophic consequences. Thus splicing is a highly complex essential step in eukaryotic gene expression. It takes place in spliceosome, which is a dynamic RNA-protein complex made of snRNPs and non-snRNP proteins. The splicing process consists of following sequential steps: spliceosome formation, the first transesterification and second transesterification reactions, release of the mature mRNA and recycling of the snRNPs. The spliceosomal components produce a complex network of RNA-RNA, RNA-protein and protein-protein interactions and spliceosome experience remodeling during each splicing cycle. Helicases are essentially required at almost each step for resolution of RNA-RNA and/or RNA-protein interactions. RNA helicases share a highly conserved helicase domain which includes the motif DExD/H in the single letter amino acid code. This article will focus on members of the DExD/H-box proteins involved specially in splicing in the malaria parasite Plasmodium falciparum.     

SR proteins induce alternative exon skipping through their activities on the flanking constitutive exons

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.     

Promotion of exon 6 inclusion in HuD pre-mRNA by Hu protein family members

The Hu RNA-binding protein family consists of four members: HuR/A, HuB, HuC and HuD. HuR expression is widespread. The other three neuron-specific Hu proteins play an important role in neuronal differentiation through modulating multiple processes of RNA metabolism. In the splicing events examined previously, Hu proteins promote skipping of the alternative exons. Here, we report the first example where Hu proteins promote inclusion of an alternative exon, exon 6 of the HuD pre-mRNA. Sequence alignment analysis indicates the presence of several conserved AU-rich sequences both upstream and downstream to this alternatively spliced exon. We generated a human HuD exon 6 mini-gene reporter construct that includes these conserved sequences. Hu protein over-expression led to significantly increased exon 6 inclusion from this reporter and endogenous HuD. Studies using truncated and mutant HuD exon 6 reporters demonstrate that two AU-rich sequences located downstream of exon 6 are important. RNAi knockdown of Hu proteins decreased exon 6 inclusion. An in vitro splicing assay indicates that Hu proteins promote HuD exon 6 inclusion directly at the level of splicing. Our studies demonstrate that Hu proteins can function as splicing enhancers and expand the functional role of Hu proteins as splicing regulators.     

Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence.     

A novel SR-related protein is required for the second step of Pre-mRNA splicing

The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF65-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF35 in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.     

Alternative splicing of CCN mRNAs …. it has been upon us

Variant CCN proteins have been identified over the past decade in several normal and pathological situations. The production of CCN truncated proteins have been reported in the case of CCN2(ctgf), CCN3(nov), CCN4(wisp-1) and CCN6(wisp-3). Furthermore, the natural CCN5 is known to miss the C-terminal domain that is present in all other members of the CCN family of proteins. In spite of compelling evidence that assign important biological activities to these truncated CCN variants, their potential regulatory functions have only recently begun to be widely accepted. The report of CCN1(cyr61) intron 3 retention in breast cancer cells now confirms that, in addition to well documented post-translational processing of full length CCN proteins, alternative splicing is to be regarded as another effective way to generate CCN variants. These observations add to a previous bulk of evidence that support the existence of alternative splicing for other CCN genes. It has become clearly evident that we need to recognize these mechanisms as a means to increase the biological diversity of CCN proteins.