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。     

HnRNP A/B蛋白D亚群的生物学功能

核不均一核糖核蛋白（heterogeneous nuclear ribonucleoproteins, hnRNPs）是一类结合DNA和RNA的核蛋白,并能在核质间穿梭. A/B型hnRNPs（heterogeneous nuclear ribonucleoproteins,hnRNP A/B）是hnRNPs中研究最为清楚的一大类别,生物信息学分析hnRNP A/B可以区分成A和D两个亚群,已鉴定的D亚群主要成员包括hnRNP AB, D和DL. hnRNP A/B的D亚群均含有2个保守的RNA结合结构域（RNA binding domain, RBD）和1个Gly富集区（glycine-rich domain, GRD）,成员间的主要区别在于N端和C端的长度和序列不同. D亚群与pre-mRNA和其它蛋白质结合成微粒系统,参与pre-mRNA的加工、稳定、核输出以及翻译过程. 此外,D亚群也能结合单链和双链DNA,参与转录起始和端粒的稳定. 因此,hnRNP A/B的 D亚群在各个阶段影响基因的表达,在神经系统发育、肿瘤的发生发展及衰老过程中发挥着多样性的功能.     

EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells

KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRAS^G13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRAS^A146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRAS^A146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRAS^G13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge.     

A Two-Layered Targeting Mechanism Underlies Nuclear RNA Sorting by the Human Exosome

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HRP-2, the Caenorhabditis elegans Homolog of Mammalian Heterogeneous Nuclear Ribonucleoproteins Q and R, Is an Alternative Splicing Factor That Binds to UCUAUC Splicing Regulatory Elements

Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.Alternative pre-mRNA splicing is a mechanism for generating multiple mRNA isoforms from a single gene. This process can allow a gene to encode for more than one protein isoform. For some genes, it is a mechanism for regulating message stability through production of alternative mRNA isoforms that are substrates for the nonsense-mediated mRNA decay pathway (1). The majority of human genes undergo alternative splicing (2), and the process can be regulated in tissue-specific and developmental stage-specific manners. Current models propose that cis elements on the pre-mRNA, in exons and introns, serve as recognition sites for trans-acting protein factors that bind to the pre-mRNA and regulate assembly of the splicing machinery, thus regulating splice site choice (3).In recent years, a number of groups have employed bioinformatics techniques to identify cis splicing regulatory elements (4). These techniques include using multiple interspecies sequence alignments to identify conserved intronic regions, identification of short sequences in exons that are bounded by weak consensus splice sites, and identification of common intronic sequences flanking similarly regulated alternative exons (5–9). These efforts have added many new sequences to the list of known and potential splicing regulators. The identification of the protein factor partners for these sequences will be important for understanding their function in alternative splicing regulation.Experimental approaches have identified alternative splicing factors that interact with specific cis elements (10), but the number of trans factors discovered still lags behind the number of newly identified cis element partners. Some examples of well-characterized cis element/trans-acting factor interactions include the NOVA K homology domain splicing factor binding to the sequence UCAY (11), the FOX splicing factors binding to the sequence UGCAUG (12–14), and hnRNP³ F/H proteins binding to the sequence GGGG (15, 16). By using cross-linking immunoprecipitation followed by large scale sequencing, entire catalogs of RNAs that the splicing factors NOVA, SF2/ASF, and FOX2 bind to in vivo have been determined (17–19). These approaches have led to models for how the proteins binding to their cis regulatory elements may alter splicing. These models include a role for the relative position of a cis element to an alternative cassette exon in determining alternative exon inclusion or skipping (18, 19).In a previous bioinformatics analysis of evolutionarily conserved intronic sequences flanking alternatively spliced exons, we identified the hexamer sequence UCUAUC as a novel splicing regulatory element (8). UCUAUC is found flanking both sides of alternative exon 16 of the unc-52 gene of Caenorhabditis elegans. Genetic analysis of a class of viable unc-52 mutants led to the discovery that exons 16–18 are alternative cassette exons and that every combination of skipping and inclusion of these three exons occurs (20). This splicing is regulated by the alternative splicing factor MEC-8 (21). Fig. 1A shows a schematic diagram of the alternatively spliced region of unc-52, with the MEC-8-enhanced alternative splicing events indicated. Using an unc-52 splicing reporter trans gene containing alternative exons 15–19, we previously reported that alternative splicing is regulated by the intronic motif UCUAUC in the intron downstream of exon 16 (8). In addition we showed that this element works cooperatively with a UGCAUG hexamer (the consensus FOX-1-binding site) in the upstream intron to regulate alternative splicing (8).Open in a separate windowFIGURE 1.RNA affinity chromatography identifies HRP-2 as binding to UCUAUC elements. A, schematic representation of the alternatively spliced region of unc-52 (adapted from Ref. 21). The alternative splicing events promoted by MEC-8 are indicated by bold lines. The lines next to introns 15 and 16 are the sites of the UCUAUC elements in those introns whose sequences were used in the RNA affinity chromatography. B, table showing sequences of RNAs immobilized to beads in the RNA affinity chromatography experiment. C, Coomassie-stained SDS-PAGE analysis of RNA affinity chromatography. C. elegans embryo extract was incubated with the different immobilized RNA substrates listed on top of the gel. Proteins identified by mass spectrometry are listed to the right of the gel, with arrows pointing to coincident protein bands. D, the left panel shows the silver stain result for the RNA affinity chromatography experiment. Each lane represents a different immobilized substrate, as indicated above. The band corresponding to HRP-2 is indicated by an arrow. The right panel is an immunoblot of the same gel using anti-HRP-2 polyclonal antibody. E, anti-HRP-2 immunoblot of an RNA affinity chromatography experiment for the indicated substrates.In this study, we report the results of a biochemical identification of a protein factor from C. elegans that binds to the UCUAUC intronic splicing regulatory element. We transcribed different short RNA sequences containing the UCUAUC element in its native intronic context, or as part of a repeating unit, and immobilized these onto agarose beads. After passing embryo extracts across these beads, we found that the protein HRP-2, the C. elegans homolog of the mammalian hnRNP Q/R proteins, binds to this sequence with high affinity. By using RNAi to reduce the level of HRP-2 in worms, we observed changes in alternative splicing of unc-52 and lin-10, two genes that contain UCUAUC elements in introns flanking alternative exons. We propose that HRP-2 is an alternative splicing factor that works through the UCUAUC intronic elements to regulate alternative splicing.     

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.     

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.