Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.     

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.     

Heterogeneous nuclear ribonucleoproteins F and H/H' show differential expression in normal and selected cancer tissues

The heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H/H', containing the quasi-RNA recognition motif (qRRM) domains, are implicated in several steps of pre-mRNA processing and in cellular differentiation. We have compared a set of tissues and found striking differences in their levels of expression as well as in the nuclear versus the cytoplasmic distribution. Generally, hnRNP F is broadly expressed in many tissues with extremely strong expression in the prostate gland while hnRNP H/H' shows a more restricted degree of expression with low expression in some tissues, for example, liver, exocrine acini of the pancreas, thyroid gland and heart. At the cellular level, hnRNP F is, with few exceptions, predominantly expressed in the cytoplasm while hnRNP H/H' is more abundant in the nuclei. A quite pronounced heterogeneous expression pattern is seen in the proximal tubules of the kidney where hnRNP F is present at moderate cytoplasmic levels while hnRNP H/H' is undetectable, whereas both proteins are more evenly expressed in distal tubules and collecting ducts. Generally, tumor tissues reveal a broad expression of hnRNP F in the nuclei as well as in the cytoplasm while hnRNP H/H' is expressed at higher levels in the nuclei than in the cytoplasm. Up-regulation of hnRNP H/H' is found in a few tissues that normally express low cytoplasmic levels of hnRNP H/H', for example, adenocarcinoma of the pancreas, hepatocellular carcinoma and gastric carcinoma. hnRNP F is down-regulated in hepatocellular carcinoma and up-regulated in gastric carcinoma. The present study indicates the important potential role of this subset of hnRNPs on the gene expression in many tissues.     

Direct interaction between hnRNP‐M and CDC5L/PLRG1 proteins affects alternative splice site choice

Heterogeneous nuclear ribonucleoprotein‐M (hnRNP‐M) is an abundant nuclear protein that binds to pre‐mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP‐M and the human spliceosome proteins cell division cycle 5‐like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat‐shock stress response. A central region in hnRNP‐M is required for interaction with CDC5L/PLRG1. hnRNP‐M affects both 5′ and 3′ alternative splice site choices, and an hnRNP‐M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno‐E1A mini‐gene substrate.     

姜黄素诱导人成骨肉瘤MG-63细胞凋亡过程中hnRNP A2/B1表达与定位

姜黄素(curcumin)诱导处理的人成骨肉瘤MG-63细胞,在光镜和电镜观察细胞凋亡的基础上,对hnRNP A2/B1在核基质中存在、分布及其与凋亡相关基因产物在MG-63细胞中的共定位关系进行了研究.经姜黄素处理后,细胞出现染色质凝聚、细胞核固缩、凋亡小体等典型的细胞凋亡形态特征;双向凝胶电泳和质谱鉴定结果显示,hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,在姜黄素处理后细胞核基质蛋白中表达下调.蛋白质印迹杂交结果,证实hnRNP A2/B1在姜黄素处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化.免疫荧光显微镜观察显示,hnRNP A2/B1定位于MG-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平变化.激光扫描共聚焦显微镜的观察结果显示,hnRNP A2/B1在MG-63细胞凋亡过程中与Bax、Bcl-2、Fas和p53等基因产物具有共定位关系,且其共定位区域发生了变化.研究结果证实了hnRNP A2/B1定位于核基质纤维上,是一种核基质蛋白,在姜黄素诱导人成骨肉瘤MG-63凋亡过程中的表达与分布变化及其与凋亡相关基因的关系显然对MG-63细胞凋亡具有重要影响,这为深入揭示肿瘤细胞凋亡的机制提供了重要科学依据和深入探索的新方向.     

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.     

The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)-rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK(3/6)-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.     

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.     

Expression profile and interactions of hnRNP A3 within hnRNP/mRNP complexes in mammals

The hnRNP A/B family contains abundant nuclear proteins with major roles in alternative splicing and the ability for nucleo-cytoplasmic shuttling. Compared to the best known members of this family (hnRNP A1, A2/B1), hnRNP A3 is a relatively less known protein. We report herein immunochemical studies with the hnRNP A3 isoforms (A3a and A3b) that provided evidence for species-specific expression. The unspliced A3a was found in human and murine cells, while the spliced A3b was a unique and abundant isoform in mouse/rat. In addition, a tissue-specific variation was observed in mice, as the brain was the only tissue found to overexpress hnRNP A3a. Both hnRNP A3a and A3b were able to stably associate with immunoselected hnRNP and mRNP complexes. Use of the auxiliary domain of hnRNP A3 in pull-down assays on human cell extracts revealed its unique ability to form a network of interactions not only with other A/B proteins but also with additional hnRNPs. All interactions, except those of hnRNP A1, were highly enhanced by previous RNase A digestion of the extracts. Our findings revealed novel characteristics of hnRNP A3 and supported its extensive involvement in the many aspects of mRNA maturation processes along with the other hnRNP A/B proteins.