hnRNP A2/B1在人永生化表皮HaCaT细胞凋亡过程中的表达及其调控作用研究

该文以姜黄素诱导人永生化表皮HaCaT细胞凋亡为基础,对hnRNP A2/B1在核基质中的存在、分布及其与细胞凋亡相关基因产物的共定位及相互作用关系进行了研究。蛋白质印迹结果显示,hnRNP A2/B1存在于HaCaT细胞核基质蛋白组分中,在经过姜黄素处理后,表达下调;激光共聚焦显微镜观察显示,hnRNP A2/B1在HaCaT细胞中分别与Fas、p53和Bax等基因产物具有共定位关系,姜黄素处理后其共定位区域出现由核膜或核仁向胞质转移的趋势。GST pull-down实验证实,hnRNPA2/B1分别与Fas、p53和Bax有直接相互作用关系。结果表明,hnRNPA2/B1作为一种核基质蛋白,通过与细胞凋亡相关基因产物的相互作用参与HaCaT细胞的凋亡诱导调控过程,这对深入认识核基质蛋白在细胞凋亡过程中的调控机制具有重要意义。     

Dual localization of the human gene encoding hnRNP I/PTB protein to chromosomes 19p13.3 and 14q23

A cDNA probe representative of the human hnRNP I/PTB gene was used to perform fluorescence in situ hybridization (FISH) on metaphases of human chromosomes. A new localization was found on band 19p13.3 in addition to the previously reported localization to band 14q23. Identical results were obtained when FISH analysis was repeated with probes covering different parts of the hnRNP I cDNA clone. This supported the notion that most, if not all, of the sequences of the different parts of this clone are present on both chromosomes. Moreover, Southern blot analysis of DNAs from interspecies somatic hybrids containing chromosomes 19 and 14 revealed that the whole hnRNP I cDNA probe generated very similar patterns in each hybrid DNA. These data suggest that two closely related copies of the hnRNP I gene exist in the human genome. Received: 19 January 1996 / Revised: 9 March 1996     

Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

In myotonic dystrophy (DM1), both inactivation of muscleblind proteins and increased levels of CUG-BP1 are reported. These events have been shown to contribute independently to aberrant splicing of a subset RNAs. We demonstrate that steady-state levels of the splice regulator, hnRNP H, are elevated in DM1 myoblasts and that increased hnRNP H levels in normal myoblasts results in the inhibition of insulin receptor (IR) exon 11 splicing in a manner similar to that observed in DM1. In normal myoblasts, overexpression of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1, which is required to maximally inhibit IR exon 11 inclusion. Elevated levels of MBNL1 show RNA-independent interaction with hnRNP H and dampen the inhibitory activity of increased hnRNP H levels on IR splicing in normal myoblasts. In DM1 myoblasts, overexpression of MBNL1 in conjunction with si-RNA mediated depletion of hnRNP H contributes to partial rescue of the IR splicing defect. These data demonstrate that coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 dictate IR splicing in normal and DM1 myoblasts.     

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.     

hnRNP K的结构及功能

核内不均一性核糖核蛋白K（heterogeneous nuclear ribonucleoprotein K,hnRNP K）最早在hnRNA加工过程中被发现,属于hnRNP家族的一员。研究表明hnRNPK的主要功能结构为3个引导DNA—RNA连接的KH域和一个独特的KI域。hnRNP K不仅能够通过依赖CT元件的途径或不依赖CT元件的途径在转录水平上对基因表达进行调控,还能够通过自身的磷酸化,改变mRNA的翻译效率,以及调控基因翻译及转导胞内信号。此外,hnRNP K与肿瘤发生和转移的关系也是近年来的研究热点。hnRNP K被发现在许多肿瘤组织中高表达,主要通过调控与细胞增殖有关的基因表达而影响肿瘤的发生发展,同时它与肿瘤细胞的扩散转移也有关。     

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.     

Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation.     

PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication.     

Purification of human telomerase complexes identifies factors involved in telomerase biogenesis and telomere length regulation

The identities and roles of proteins associated with human telomerase remain poorly defined. To gain insight, we undertook an affinity purification of endogenously assembled human telomerase complexes. We show that specific subsets of H/ACA, Sm, and hnRNP proteins associate with active and inactive telomerase RNPs, while two NTPase proteins associate preferentially with active enzyme. All three core H/ACA-motif binding proteins are telomerase holoenzyme components essential for RNP accumulation. On the other hand, telomerase RNPs lacking interaction with Sm proteins or hnRNP C remain fully functional for telomere elongation. Curiously, overexpression of either associated hnRNP protein (hnRNP C and hnRNP U) or either NTPase protein (NAT10 and GNL3L) induced telomere shortening. Our findings suggest that endogenous human telomerase complexes are more heterogeneous than those of single-celled eukaryotes, have predominantly shared rather than telomerase-specific proteins, and make numerous regulatory interactions.     

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.     

HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.     

Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 — GWbs marker protein and TIA-1 stress granule component.