Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins.

hnRNP A1 is a pre-mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5' splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP-1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA-protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre-mRNA binding or RNA annealing activity. The C-terminal Gly-rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly-rich domain, binds more strongly to pre-mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre-mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

hnRNP A1 relocalization to the stress granules reflects a role in the stress response

hnRNP A1 is a nucleocytoplasmic shuttling protein that is involved in many aspects of mRNA metabolism. We have previously shown that activation of the p38 stress-signaling pathway in mammalian cells results in both hyperphosphorylation and cytoplasmic accumulation of hnRNP A1, affecting alternative splicing regulation in vivo. Here we show that the stress-induced cytoplasmic accumulation of hnRNP A1 occurs in discrete phase-dense particles, the cytoplasmic stress granules (SGs). Interestingly, mRNA-binding activity is required for both phosphorylation of hnRNP A1 and localization to SGs. We also show that these effects are mediated by the Mnk1/2 protein kinases that act downstream of p38. Finally, depletion of hnRNP A1 affects the recovery of cells from stress, suggesting a physiologically significant role for hnRNP A1 in the stress response. Our data are consistent with a model whereby hnRNP A1 recruitment to SGs involves Mnk1/2-dependent phosphorylation of mRNA-bound hnRNP A1.     

Hypophosphorylated ASF/SF2 binds TAP and is present in messenger ribonucleoproteins

Serine/arginine-rich proteins (SR proteins) function in precursor mRNA (pre-mRNA) splicing and may also act as adaptors for mRNA export. SR proteins are dynamically phosphorylated in their RS domain, and differential phosphorylation modulates their splicing activity and subcellular localization. In this study, we investigated the influence of phosphorylation on the function of SR proteins in events occurring during mRNA maturation. Immunoprecipitation experiments showed that the mRNA export receptor TAP associates preferentially with the hypophosphorylated form of shuttling SR proteins, including ASF/SF2. Overexpression of ASF induced subnuclear relocalization of TAP to SR protein-enriched nuclear speckles, suggesting their interaction in vivo. Moreover, the ASF found in a nucleoplasmic fraction rich in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes is hyperphosphorylated, whereas mature messenger RNP (mRNP)-bound ASF is hypophosphorylated. Therefore, hypophosphorylation of ASF in mRNPs coincides with its higher affinity for TAP, suggesting that dephosphorylation of ASF promotes both its incorporation into mRNPs and recruitment of TAP for mRNA export. Thus, the phosphorylation state of RS domains may modulate the function of mammalian shuttling SR proteins during mRNA maturation or export.     

MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence.     

hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing.

Splicing of the human immunodeficiency virus type 1 (HIV-1) pre-mRNA must be inefficient to provide a pool of unspliced messages which encode viral proteins and serve as genomes for new virions. Negative cis-regulatory elements (exonic splicing silencers or ESSs) are necessary for HIV-1 splicing inhibition. We demonstrate that heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A and B group are trans-acting factors required for the function of the tat exon 2 ESS. Depletion of hnRNP A/B proteins from HeLa cell nuclear extract activates splicing of tat exon 2 pre-mRNA substrate. Splicing inhibition is restored by addition of recombinant hnRNP A/B proteins to the depleted extract. A high-affinity hnRNP A1-binding sequence can substitute functionally for the ESS in tat exon 2. These results demonstrate that hnRNP A/B proteins are required for repression of HIV-1 splicing.     

Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch

A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE(16)) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre-mRNAs showed that CE(16) can repress splicing of upstream introns, and that mutagenesis or replacement of CE(16) can relieve this inhibition. An affinity selection assay with biotinylated CE(16) RNA demonstrated specific binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-specific activation of the E16 splicing switch in concert with a dramatic and specific down-regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre-mRNA splicing.     

ICP27 interacts with SRPK1 to mediate HSV splicing inhibition by altering SR protein phosphorylation

Infection with some viruses can alter cellular mRNA processing to favor viral gene expression. We present evidence that herpes simplex virus 1 (HSV-1) protein ICP27, which contributes to host shut-off by inhibiting pre-mRNA splicing, interacts with essential splicing factors termed SR proteins and affects their phosphorylation. During HSV-1 infection, phosphorylation of several SR proteins was reduced and this correlated with a subnuclear redistribution. Exogenous SR proteins restored splicing in ICP27-inhibited nuclear extracts and SR proteins isolated from HSV-1-infected cells activated splicing in uninfected S100 extracts, indicating that inhibition occurs by a reversible mechanism. Spliceosome assembly was blocked at the pre-spliceosomal complex A stage. Furthermore, we show that ICP27 interacts with SRPK1 and relocalizes it to the nucleus; moreover, SRPK1 activity was altered in the presence of ICP27 in vitro. We propose that ICP27 modifies SRPK1 activity resulting in hypophosphorylation of SR proteins impairing their ability to function in spliceosome assembly.     

Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.     

Paraquat Modulates Alternative Pre-mRNA Splicing by Modifying the Intracellular Distribution of SRPK2

Paraquat (PQ) is a neurotoxic herbicide that induces superoxide formation. Although it is known that its toxic properties are linked to ROS production, the cellular response to PQ is still poorly understood. We reported previously that treatment with PQ induced genome-wide changes in pre-mRNA splicing. Here, we investigated the molecular mechanism underlying PQ-induced pre-mRNA splicing alterations. We show that PQ treatment leads to the phosphorylation and nuclear accumulation of SRPK2, a member of the family of serine/arginine (SR) protein-specific kinases. Concomitantly, we observed increased phosphorylation of SR proteins. Site-specific mutagenesis identified a single serine residue that is necessary and sufficient for nuclear localization of SRPK2. Transfection of a phosphomimetic mutant modified splice site selection of the E1A minigene splicing reporter similar to PQ-treatment. Finally, we found that PQ induces DNA damage and vice versa that genotoxic treatments are also able to promote SRPK2 phosphorylation and nuclear localization. Consistent with these observations, treatment with PQ, cisplatin or γ-radiation promote changes in the splicing pattern of genes involved in DNA repair, cell cycle control, and apoptosis. Altogether, our findings reveal a novel regulatory mechanism that connects PQ to the DNA damage response and to the modulation of alternative splicing via SRPK2 phosphorylation.     

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase-dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for 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.     

核内不均一核糖核蛋白在前体mRNA加工中的功能

核内不均一核糖核蛋白(hnRNP)是一类存在于真核生物体内具有类似结构特征的高丰度RNA结合蛋白,一般均匀分布在核内。多种hnRNP具有多样的功能,参与从转录调节,前体mRNA剪接,mRNA输出到mRNA降解等多种生物过程,从而进行基因表达调控。现着重介绍hnRNP在前体mRNA加工过程(加帽,剪接,加尾,输出,选择性降解)中的功能及研究进展。     

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.     

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.