Role for RNA-binding proteins implicated in pathogenic development of Ustilago maydis

Ustilago maydis causes smut disease on corn. Successful infection depends on a number of morphological transitions, such as pheromone-dependent formation of conjugation tubes and the switch to filamentous dikaryotic growth, as well as different types of mycelial structures during growth within the host plant. In order to address the involvement of RNA-binding proteins during this developmental program, we identified 27 open reading frames from the genome sequence encoding potential RNA-binding proteins. They exhibit similarities to RNA-binding proteins with Pumilio homology domains (PUM), the K homology domain (KHD), the double-stranded RNA binding motif (DSRM), and the RNA recognition motif (RRM). For 18 of these genes, we generated replacement mutants in compatible haploid strains. Through analysis of growth behavior, morphology, cyclic AMP response, mating, and pathogenicity, we identified three candidates with aberrant phenotypes. Loss of Khd1, a K homology protein containing three KHDs, resulted in a cold-sensitive growth phenotype. Deletion of khd4 encoding a protein with five KHDs led to abnormal cell morphology, reduced mating, and virulence. rrm4Delta strains were affected in filamentous growth and pathogenicity. Rrm4 is an RRM protein with a so far unique domain organization consisting of three N-terminal RRMs as well as a domain found in the C terminus of poly(A)-binding proteins. These results indicate a role for RNA-binding proteins in regulation of morphology as well as in pathogenic development in U. maydis.     

Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR.

The porcine group C rotavirus (Cowden strain) NSP3 protein (the group C equivalent of the group A gene 7 product, formerly called NS34) shares homology with known double-stranded RNA-binding proteins, such as the interferon-induced, double-stranded RNA-dependent protein kinase PKR. A clone of NSP3, expressed both in vitro and in COS-1 cells, led to the synthesis of minor amounts of a product with an M(r) of 45,000 (the expected full-length M(r) of NSP3) and major amounts of products with M(r)s of 38,000 and 8,000. Restriction enzyme digestion analysis prior to expression in vitro and amino-terminal sequence analysis suggest that the products with M(r)s of 38,000 and 8,000 are cleavage products of the protein with an M(r) of 45,000. The full-length protein and the product with an M(r) of 8,000, both of which contain the motif present in double-stranded RNA-binding proteins, bound specifically to double-stranded RNA. The products with M(r)s of 45,000 and 8,000 were also detected in Cowden strain-infected MA104 cells. NSP3 products expressed in COS-1 cells were capable of inhibiting activation of the double-stranded RNA-dependent protein kinase similar to other double-stranded RNA-binding proteins, and NSP3 products expressed in HeLa cells were capable of rescuing the replication of an interferon-sensitive deletion mutant of vaccinia virus.     

Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus

Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.     

Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen.

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.     

Characterization of the bovine herpesvirus 4 major immediate-early transcript.

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.     

蚕豆萎蔫病毒2号板蓝根分离株全基因组序列测定与分析

板蓝根(Isatidis Radix)病毒病害的发生已对其产量和品质造成了严重影响。因此,建立一套灵敏、快速、有效的板蓝根病毒病害检测手段十分重要。本研究利用双链RNA(double-stranded RNA,dsRNA)和非序列依赖PCR扩增(sequence-independent amplification,SIA)等技术对感病板蓝根进行鉴定,确定其被蚕豆萎蔫病毒2号(Broad bean wilt virus 2, BBWV2)所侵染。为明确BBWV2板蓝根分离物(BBWV2-IR)的进化关系,对其全基因组进行序列扩增与分析,获得其RNA1序列全长为5 955 bp,RNA2序列全长为3 602 bp,分别编码由1 870和1 064个氨基酸组成的多聚蛋白质。序列比对发现,BBWV2-IR RNA1与BBWV2-Am分离物的同源性最高,RNA2与BBWV2-SN分离物的同源性最高。全基因组变异情况分析表明,BBWV2-IR RNA1和RNA2分别与其同源性最高的株系存在多个氨基酸变异位点。系统进化分析表明,BBWV2-IR RNA1与BBWV2-Am RNA1聚为一簇,RNA2与BBWV2-SN RNA2聚为一簇,亲缘性最近。本研究获得了BBWV2板蓝根分离株的全基因组序列,并明确其在进化过程中的地位和区域变化情况,为进一步研究BBWV2-IR致病性变异提供一定的理论基础。     

A trinucleotide repeat-associated increase in the level of Alu RNA-binding protein occurred during the same period as the major Alu amplification that accompanied anthropoid evolution.

Nearly 1 million Alu elements in human DNA were inserted by an RNA-mediated retroposition-amplification process that clearly decelerated about 30 million years ago. Since then, Alu sequences have proliferated at a lower rate, including within the human genome, in which Alu mobility continues to generate genetic variability. Initially derived from 7SL RNA of the signal recognition particle (SRP), Alu became a dominant retroposon while retaining secondary structures found in 7SL RNA. We previously identified a human Alu RNA-binding protein as a homolog of the 14-kDa Alu-specific protein of SRP and have shown that its expression is associated with accumulation of 3'-processed Alu RNA. Here, we show that in early anthropoids, the gene encoding SRP14 Alu RNA-binding protein was duplicated and that SRP14-homologous sequences currently reside on different human chromosomes. In anthropoids, the active SRP14 gene acquired a GCA trinucleotide repeat in its 3'-coding region that produces SRP14 polypeptides with extended C-terminal tails. A C-->G substitution in this region converted the mouse sequence CCA GCA to GCA GCA in prosimians, which presumably predisposed this locus to GCA expansion in anthropoids and provides a model for other triplet expansions. Moreover, the presence of the trinucleotide repeat in SRP14 DNA and the corresponding C-terminal tail in SRP14 are associated with a significant increase in SRP14 polypeptide and Alu RNA-binding activity. These genetic events occurred during the period in which an acceleration in Alu retroposition was followed by a sharp deceleration, suggesting that Alu repeats coevolved with C-terminal variants of SRP14 in higher primates.     

Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI.

Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity.     

汉坦病毒疫苗株Z10与Z37重组核蛋白NP的表达纯化及其特异结合基因组末端反向重复序列的功能研究

汉坦病毒是引起肾综合征出血热(HFRS)和汉坦病毒型肺炎综合征(HPS)的主要病原体.其基因组由三节段的单股负链RNA组成,即S、M与L基因片段.汉坦病毒基因组的一个重要特点是每个基因片段的两个末端都有一段长18个核苷酸的高度保守的反向重复序列,互补可形成双链发夹结构,并且这一特点为不同型病毒所共有.为了研究该基因组末端保守的反向重复序列的功能,首先构建了汉坦病毒中国疫苗株Z10(汉滩型)及Z37(汉城型)的核蛋白原核表达载体,并在大肠杆菌中高效表达.经NI-NAT亲和柱和HiPrep16/10 DEAE离子交换柱液相色谱(FPLC)二步提纯,获得高纯度的重组核蛋白,并分别以胰蛋白酶消化后,用Western blotting进行区分和鉴定.以T4 DNA激酶同位素标记一对人工合成互补的18个核苷酸反向重复序列,制备双链探针.然后将该探针与纯化的Z10、Z37株的核蛋白NP进行非变性凝胶电泳迁移改变实验(EMSA)后发现,重组的Z10、Z37株的核蛋白NP,在体外均可特异地结合其基因组末端反向重复序列形成的双链探针.该结果表明,汉坦病毒基因组末端的反向重复序列是核蛋白重要结合位点,这对理解汉坦病毒核蛋白功能以及病毒复制过程中病毒粒子的包装机制有重要的意义.     

RPC40, a unique gene for a subunit shared between yeast RNA polymerases A and C

Yeast RNA polymerases A and C share an approximately equal to 40 kd subunit. We have identified, sequenced, and mutagenized in vitro the AC40 subunit gene. The RPC40 gene is unique in the yeast genome and is required for cell viability. This gene contains an open reading frame encoding a 37.6 kd protein having no significant homology with bacterial RNA polymerase subunits. The promoter region contains a 19 bp sequence also present in the largest subunit of RNA polymerase C. It also contains a well-conserved RPG box, a sequence found in the promoter region of many genes encoding the translational apparatus. A novel, plasmid-shuffling method was developed to isolate a large number of RPC40 ts mutants. One of these, ts4, was shown to be defective in the synthesis of RNA polymerases A and C at the restrictive temperature. In contrast, RNA polymerase B was made normally.     

RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.     

Cloning and characterization of the mouse Interleukin Enhancer Binding Factor 3 (Ilf3) homolog in a screen for RNA binding proteins

In a screen for RNA-binding proteins expressed during murine spermatogenesis, we have identified a cDNA that encodes a protein of 911 amino acids that contains two copies of the double-stranded RNA-binding motif and has 80% identity with human Interleukin Enhancer Binding Factor 3 (ILF3). Linkage and cytogenetic analyses localized the Ilf3 cDNA to a portion of mouse Chr 9, which shows conserved synteny with a region of human Chr 19 where the human ILF3 gene had been previously localized, supporting that we had cloned the murine homolog of ILF3. Northern analysis indicated the Ilf3 gene is ubiquitously expressed in mouse adult tissues with high levels of expression in the brain, thymus, testis, and ovary. Polyclonal antibodies detected multiple protein species in a subset of the tissues expressing Ilf3 RNA. Immunoreactive species are present at high levels in the thymus, testis, ovary, and the spleen to a lesser extent. The high degree of sequence similarity between the mouse ILF3 protein and other dsRNA binding motif-containing proteins suggests a role in RNA metabolism, while the differential expression indicates the mouse ILF3 protein predominantly functions in tissues containing developing lymphocyte and germ cells. Received: 21 October 1998 / Accepted: 15 January 1999     

水稻矮缩病毒第11号组分基因序列和编码蛋白的功能分析

水稻矮缩病毒(Rice Dwarf Virus-RDV)广泛分布于中国、日本及东南亚地区,侵染水稻和禾本科其它一些作物,是造成水稻减产的主要原因之一,对农作物危害极大。RDV属于呼肠孤病毒科(Re-oviridae)中的植物呼肠孤病毒属(Phytoreovirus)成员,其病毒粒子直径70nm,为20面体,有双层