Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the upper and lower motor neurons. 5-10% of cases are genetically inherited, including ALS type 20, which is caused by mutations in the hnRNPA1 gene. The goals of this work are to analyze the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on hnRNPA1 protein function, to model the complete tridimensional structure of the protein using computational methods and to assess structural and functional differences between the wild type and its variants through Molecular Dynamics simulations. nsSNP, PhD-SNP, Polyphen2, SIFT, SNAP, SNPs&GO, SNPeffect and PROVEAN were used to predict the functional effects of nsSNPs. Ab initio modeling of hnRNPA1 was made using Rosetta and refined using KoBaMIN. The structure was validated by PROCHECK, Rampage, ERRAT, Verify3D, ProSA and Qmean. TM-align was used for the structural alignment. FoldIndex, DICHOT, ELM, D2P2, Disopred and DisEMBL were used to predict disordered regions within the protein. Amino acid conservation analysis was assessed by Consurf, and the molecular dynamics simulations were performed using GROMACS. Mutations D314V and D314N were predicted to increase amyloid propensity, and predicted as deleterious by at least three algorithms, while mutation N73S was predicted as neutral by all the algorithms. D314N and D314V occur in a highly conserved amino acid. The Molecular Dynamics results indicate that all mutations increase protein stability when compared to the wild type. Mutants D314N and N319S showed higher overall dimensions and accessible surface when compared to the wild type. The flexibility level of the C-terminal residues of hnRNPA1 is affected by all mutations, which may affect protein function, especially regarding the protein ability to interact with other proteins.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a host factor required for dengue virus and Junín virus multiplication.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are cellular factors involved in the replication of several viruses. In this study we analyzed the expression and intracellular localization of hnRNP A2 and hnRNP K in cell cultures infected with two viruses that cause human hemorrhagic fevers: dengue virus type 2 (DENV-2) and Junín virus (JUNV). We determined that DENV-2 promoted the cytoplasmic translocation of hnRNP K and to a lesser extent of hnRNP A2, meanwhile, JUNV infection induced an increase in hnRNP K cytoplasmic localization whereas hnRNP A2 remained mainly in the nucleus of infected cells. Both hnRNP K and hnRNP A2 were localized predominantly in the nucleus of JUNV persistently-infected cells even after superinfection with JUNV indicating that persistent infection does not alter nucleo-cytoplasmic transport of these hnRNPs. Total levels of hnRNP K expression were unaffected by DENV-2 or JUNV infection. In addition we determined, using small interfering RNAs, that hnRNP K knockout inhibits DENV-2 and JUNV multiplication. Our results indicate that DENV-2 and JUNV induce hnRNP K cytoplasmic translocation to favor viral multiplication.

Changes in splicing factor expression are associated with advancing age in man.

Human ageing is associated with decreased cellular plasticity and adaptability. Changes in alternative splicing with advancing age have been reported in man, which may arise from age-related alterations in splicing factor expression. We determined whether the mRNA expression of key splicing factors differed with age, by microarray analysis in blood from two human populations and by qRT-PCR in senescent primary fibroblasts and endothelial cells. Potential regulators of splicing factor expression were investigated by siRNA analysis. Approximately one third of splicing factors demonstrated age-related transcript expression changes in two human populations. Ataxia Telangiectasia Mutated (ATM) transcript expression correlated with splicing factor expression in human microarray data. Senescent primary fibroblasts and endothelial cells also demonstrated alterations in splicing factor expression, and changes in alternative splicing. Targeted knockdown of the ATM gene in primary fibroblasts resulted in up-regulation of some age-responsive splicing factor transcripts. We conclude that isoform ratios and splicing factor expression alters with age in vivo and in vitro, and that ATM may have an inhibitory role on the expression of some splicing factors. These findings suggest for the first time that ATM, a core element in the DNA damage response, is a key regulator of the splicing machinery in man.

Involvement of hnRNP A1 in the matrix metalloprotease-3-dependent regulation of Rac1 pre-mRNA splicing.

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer.

Differential effect of acute and persistent Junin virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B.

Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.

Identification of multiple differentially expressed messenger RNAs in normal and pathological trophoblast.

In an attempt to assess the molecular basis of phenotypic alterations present in the gestational trophoblastic diseases (GTDs) and to identify genes whose expression is specifically associated to these placental proliferative disorders we performed differential display techniques. Initially 19 candidate gene fragments were identified and differential expression was confirmed in eight of these fragments by Northern blot analysis. At the mRNA level ribosomal L26 (rL26), ribosomal L27 (rL27), a new Krüppel type zinc finger protein and TIS11d were preferentially expressed in normal early placenta (NEP) relative to complete hydatidiform mole (CHM), persistent gestational trophoblastic disease (PGTD) and choriocarcinoma JEG-3 cell line. In contrast, heterogeneous ribonucleoprotein A1 (hnRNPA1), the ferritin light chain mRNA, and the uncharacterized protein KIAA0992 were predominantly expressed in JEG-3 cell line. Finally, decorin, a prototype member of an expanding family of small leucine-rich proteoglycans, showed high expression in CHM. In addition we demonstrated by immunohistochemistry analysis that increased decorin mRNA in CHM reflected a genuine augmentation in average steady state mRNA levels within cells. Taken together, these findings provide several interesting candidates for regulation of tumorigenic expression as well as early placentation development, including those involved in protein synthesis (rL26 and rL27), metabolism (ferritin light chain), intercellular communication (decorin) and regulation of gene expression (Krüppel-like zinc finger, TIS11d and hnRNPA1). Information about such alterations in gene expression could be useful for elucidating the genetic events associated to gestational trophoblastic pathogenesis, developing new diagnostic markers, or determining novel therapeutic targets.

RNA annealing activities in HeLa nuclei.

RNA-RNA base pairing plays a critical role in the interactions between pre-mRNAs and trans-acting factors during the processing of pre-mRNAs (hnRNAs) into mRNAs, and it is likely that specific factors are required to promote the annealing of RNAs. To identify particular nuclear components that have such activity, we fractionated HeLa nucleoplasm and assayed for activity which promoted the hybridization of a pre-mRNA with an antisense RNA probe complementary to 60 nucleotides (nt) encompassing the 3' splice site. At least nine major RNA annealing activities were identified and, surprisingly, eight of these copurified partially or to homogeneity with known hnRNP proteins. The activities of three of these proteins, hnRNP A1, C1 and U, were confirmed using purified recombinant proteins. Moreover, we found that the RNA binding domain alone of hnRNP C1/C2 had significant activity, indicating that this RNA annealing may result, at least partly, from chaperone activity: a direct modulation of RNA conformation by hnRNP proteins. The finding that hnRNP proteins have strong RNA annealing activity indicates that they can profoundly affect the interactions of pre-mRNAs with trans-acting factors and suggests this to be an important function of hnRNP proteins in the processing of pre-mRNAs.