Member Of hnRNP Family Research Articles

Heterogeneous Ribonucleoprotein M Is a Splicing Regulatory Protein That Can Enhance or Silence Splicing of Alternatively Spliced Exons

Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation.

Sequence and transcriptional study ofHNRPKpseudogenes, and expression and molecular modeling analysis of hnRNP K isoforms

The heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a large family of proteins that play important roles in telomere biogenesis, DNA repair, cellular signaling, and the regulation of expression at both the transcriptional and translational levels. One of the most extensively studied hnRNP family members, hnRNP K, has been implicated in a variety of processes, including chromatin remodeling, transcription, splicing, and translation events. In this study, we analyzed processed HNRPK pseudogenes (HNRPK psi1-psi4) and coding sequences. HNRPK pseudogenes are apparently nonfunctional, and psi1 might correspond to transcripts from an ancestral gene. Phylogenetic and sequence analyses suggest that HNRP genes arose by duplication, and that new structural and sequence features expanded the functions of hnRNPs. The expression analysis of hnRNP K isoforms showed that isoform a is expressed in normal testis and in non-small cell lung cancer (NCI-H1155 NSCLC cell line), although the shorter isoform (isoform b) is expressed in different tumor cell lines (IM9 B-lymphoblastoid, Hs578T human breast cancer epithelial, T98G human glioma cell lines). Using molecular modeling, we obtained KH1 and KH3 models, which pointed to important residues for DNA-protein binding and no structural differences between isoforms a and b. To our knowledge, this is the first phylogenetic study including vertebrate HNRP genes and HNRPK pseudogenes, and the first report comparing the KH1 and KH3 domains of isoforms a and b of the hnRNP K protein. New investigations in tumor samples must be done to validate the differential expression observed here. The results shown are important because the hnRNP K protein might represent a new target for pharmacologic intervention in virus replication and cancer.

Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf(+/)(-) mice were apparently healthy and fertile, no viable acf(-)(/)(-) mice were identified. Mutant acf(-)(/)(-) embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf(-)(/)(-) blastocysts were detectable following implantation at E4.5, and isolated acf(-)(/)(-) blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

The interaction of the hnRNP family member E1B-AP5 with p53

Adenovirus early region 1B-associated protein 5, E1B-AP5, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, was originally isolated on the basis of its ability to bind to the adenovirus 5 early region1B55K protein. Here, it has been demonstrated that E1B-AP5 interacts with mutant and wild-type p53 from human cells in pull-down assays using GST-E1B-AP5. This interaction has been confirmed by co-immunoprecipitation studies and pull-down experiments with in vitro translated E1B-AP5 and GST-p53. The binding site for E1B-AP5 has been mapped to the C-terminal region of p53. In reciprocal experiments, it has been shown that several regions of E1B-AP5 bound to p53 although it is probable that a major site of interaction is located between amino acids 395 and 732 of E1B-AP5. In reporter assays, E1B-AP5 inhibited p53 transcriptional activity although not as efficiently as the Ad5E1B55K protein. Transfection of E1B-AP5 into human tumour cells affected the cellular response to UV radiation, such that, although p53 expression was induced, little change in the level of p53-inducible genes could be observed.

Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-alpha. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.