Specific RNA-protein Complexes Research Articles

Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods.

Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.

A widely applicable and cost-effective method for specific RNA–protein complex isolation

Although methodological advances have been made over the past years, a widely applicable, easily scalable and cost-effective procedure that can be routinely used to isolate specific ribonucleoprotein complexes (RNPs) remains elusive. We describe the “Silica-based Acidic Phase Separation (SAPS)-capture” workflow. This versatile method combines previously described techniques in a cost-effective, optimal and widely applicable protocol. The specific RNP isolation procedure is performed on a pre-purified RNP sample instead of cell lysate. This combination of protocols results in an increased RNP/bead ratio and by consequence a reduced experimental cost. To validate the method, the 18S rRNP of S. cerevisiae was captured and to illustrate its applicability we isolated the complete repertoire of RNPs in A. thaliana. The procedure we describe can provide the community with a powerful tool to advance the study of the ribonome of a specific RNA molecule in any organism or tissue type.

Elucidating the RNA-Protein Interactomes of Target RNAs in Tissue.

RNA-protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA-protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA-protein complexes.

Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces

Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on invivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.

Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro.

RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

Ribo-Proteomics Approach to Profile RNA-Protein and Protein-Protein Interaction Networks.

Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses.

The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

In eukaryotes, polyadenylation of pre-mRNA 3´ end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3´ end processing machinery in this protozoan parasite.

In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer

Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.

Genomic Analyses of Musashi1 Downstream Targets Show a Strong Association with Cancer-related Processes

Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3'-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation.

Posttranscriptional regulation of albumin gene expression by branched-chain amino acids in rats with acute liver injury

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched-chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepared from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the coding region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver of galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein in p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubated with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase in albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfere with this binding and improve the translation efficiency of albumin mRNA in injured liver.

Assembly of an active group II intron-maturase complex by protein dimerization.

Group II intron-encoded proteins promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein complex. The Lactococcus lactis L1.LtrB intron encodes a maturase, LtrA, with reverse transcriptase homology and specific binding affinity for two domains of the intron RNA. The catalytically active ribonucleoprotein (RNP) has splicing, endonuclease, and reverse transcriptase activity, enabling efficient insertion of the intron sequence by a retro-homing mechanism. To determine the composition and assembly mechanism of the RNP complex, purified LtrA protein was analyzed for its ability to recognize a series of intron-derived RNAs. Equilibrium dissociation measurements show that LtrA recognizes two intronic domains, DI and DIV. However, distinct electrostatic requirements for binding imply different modes of molecular recognition in each case. Stoichiometric binding experiments show that the functional RNP complex consists of a dimeric protein species bound to a single intron RNA. Significant differences between the measured equilibrium dissociation constants and kinetically derived values suggest that conformational changes accompany assembly of the intron-maturase complex, and results of limited proteolysis and fluorescence spectroscopy experiments suggest that significant RNA-dependent structural changes within the maturase occur upon association with the intron. These results support a mutually induced fit model in which RNA-dependent conformational changes within LtrA enable stable association of the protein dimer with two independent intron domains to form a functional RNP.

Selection of RRE RNA binding peptides using a kanamycin antitermination assay.

The arginine-rich domains of several RNA-binding proteins have been shown to bind their cognate RNAs with high affinities and specificities as isolated peptides, adopting different conformations within different complexes. The sequence simplicity and structural diversity of the arginine-rich motif has made it a good framework for constructing combinatorial libraries and identifying novel RNA-binding peptides, including those targeted to the HIV Rev response element (RRE). Here we describe a modified transcription antitermination reporter assay engineered with kanamycin resistance that enables larger in vivo screens (approximately 10(9) sequences) than previously possible. We show that the assay detects only specific RNA-protein complexes, and that binders are enriched at least 300-fold per round of selection. We screened a large peptide library in which amino acids with charged, polar, and small side chains were randomly distributed within a polyarginine framework and identified a set of high affinity RRE-binding peptides. Most contain glutamine at one particular peptide position, and the best peptides display significantly higher antitermination activities than Rev or other previously described high-affinity RRE-binding peptides. The kanamycin antitermination (KAN) assay should be useful for screening relatively large libraries and thereby facilitate identification of novel RNA binders.

Why do miRNAs live in the miRNP?

Noncoding RNAs function in diverse pathways—dosage compensation, gene imprinting, transcriptional regulation, pre-mRNA splicing, and the control of mRNA translation—and they carry out these roles from within specific RNA–protein complexes that ensure each noncoding RNA is in the right cellular compartment with the appropriate proteins needed to accomplish its biochemical function. Thus, identifying the ribonucleoprotein complex (RNP) associated with a noncoding RNA gives clues to its cellular function and biochemical mechanism by revealing the proteins whose company it keeps. The discovery by Dreyfuss and coworkers that microRNAs reside in a ∼550-kD (15S) particle provides new clues toward the functions of this novel and surprisingly large class of tiny, noncoding RNAs (Mourelatos et al. 2002). The first microRNA, (miRNA) lin-4, was identified in 1993 (Lee et al. 1993). Ambros and coworkers positionally cloned the lin-4 gene, a locus required for the correct timing of development in Caenorhabditis elegans, only to find that the gene encodes no protein (Lee et al. 1993). Instead, lin-4 comprises two small noncoding RNAs, one 22 nucleotides long, and a longer form, lin-4L, that can fold into a hairpin structure. Seven years later, Ruvkun and colleagues discovered that let-7, which likewise regulates developmental timing in worms, is also a tiny, noncoding RNA (Reinhart et al. 2000). Because lin-4 and let-7 control developmental timing, they have been dubbed small temporal RNAs (stRNAs). Recently, three laboratories succeeded in cloning additional stRNA-like RNAs from worms, flies, and human cells (Lagos-Quintana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). These efforts uncovered a wealth of 19–25 nucleotide RNAs, including lin-4 and let-7, which are collectively known as miRNAs (for reviews, see Moss 2001; Ruvkun 2001; Banerjee and Slack 2002). These efforts added an additional 100 tiny RNAs to the original pair of stRNAs. As anticipated by Ambros, stRNAs and miRNAs derive from longer stem-loop precursor RNAs. Thus, the longer lin-4L is the precursor of mature lin-4. Whereas many of the new miRNAs are produced constitutively, some are temporally regulated or expressed only in specific tissues. A few appear to be transcribed in coordinately regulated operons, suggesting that they are cleaved from their stem-loop precursors from within a long, common transcript. Others are found only in the germ line or in the early embryo, in which translational control dominates the hierarchy of regulatory mechanisms. The 21–25 nucleotide size of miRNAs is remarkably similar to that of small interfering RNAs (siRNAs), the 21–25 nucleotide double-stranded RNAs that mediate RNA interference (for reviews, see Bernstein et al. 2001b; Carthew 2001; Sharp 2001; Vaucheret et al. 2001; Waterhouse et al. 2001). siRNAs are generated by the endonucleolytic cleavage of long double-stranded RNA by the multidomain RNase III enzyme, Dicer (Bernstein et al. 2001a). siRNAs are then incorporated into a ∼500-kD RNP complex, the RNA-induced silencing complex (RISC), in which they provide the specificity determinants that direct an as yet unidentified protein nuclease to cleave mRNAs complementary to the siRNA (Hammond et al. 2000). lin-4 and let-7, as well as the new miRNAs, are encoded by ∼70 nucleotide stem-loop structures (Lee et al. 1993; Pasquinelli et al. 2000), whose stems are substrates for processing by Dicer (Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001). Dicer liberates miRNAs from the larger stem-loop precursors in much the same way it generates siRNAs from long dsRNA, leaving the signature 3 hydroxyl and 5 phosphate termini of an RNase III cleavage reaction. Both siRNA and stRNA production by Dicer requires ATP, consistent with the presence of an ATP-dependent helicase domain at the amino terminus of Dicer (Zamore et al. 2000; Bernstein et al. 2001a; Hutvagner et al. 2001; Nykanen et al. 2001). Mature lin-4 and let-7 are thought to bind partially complementary sequences in the 3 UTRs of their target mRNAs (Lee et al. 1993; Reinhart et al. 2000). Unlike the binding of siRNAs, which triggers target RNA destruction, binding of the stRNA lin-4, and likely let-7, leads to translational repression of their natural mRNA targets (Olsen and Ambros 1999; Reinhart et al. 2000; Slack et al. 2000). In worms, translational repression of lin-4 and let-7 target mRNAs is required for the progression from one stage of development to the next. In addition to Dicer, two members of the PPD family of proteins, ALG-1 and ALG-2, are required for the bio1Corresponding author. E-MAIL phillip.zamore@umassmed.edu; FAX (508) 856-2003. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.992502.

Distribution and Molecular Characterization of mRNA-Binding Proteins Specific to the (U)15Region of 3′ UTR of the Mouse Catalase (Cas-1)

The 3' UTR of the mouse Cas-1 mRNA, encoding the antioxidant enzyme catalase, has a U-rich motif that is conserved across species. This motif is an active site for complex and dynamic interactions involving RNA-binding proteins. The spatial, temporal, and phylogenetic distribution of the Cas-1 3'-UTR U-rich motif-specific RNA-binding proteins was evaluated by gel mobility shift and UV cross-linking assays. The specific RNA-protein complexes were observed in mouse tissue homogenates representing developmental stages as early as day 10 pc and ranged in molecular weight from approximately 38 kDa to approximately 52 kDa. These mRNA-protein complexes appeared in all vertebrate species examined (human, mouse, rat, dog, rabbit, chicken, fish, and frog) but not in insects. The approximately 38-kDa protein was the most prominent protein in vertebrates. The cDNA sequence of the mouse approximately 38-kDa protein was obtained by purification of the protein, microsequencing, and RT-PCR. The resulting 456-nt sequence, representing the partial internal cDNA sequence, and its deduced amino acid sequence were similar to the RNA recognition motif (RRM) of a protein superfamily, implicated in splicing, stability, localization, and translation of RNAs. Although the results suggest that cis element-binding activity could be a cytoplasmic regulator of Cas-1 mRNA metabolism, the significance of this binding remains to be determined.

A Conserved 3′-Untranslated Element Mediates Growth Regulation of DNA Methyltransferase 1 and Inhibits Its Transforming Activity

Ectopic expression of DNA methyltransferase 1 (DNMT1) has been proposed to play an important role in cancer. dnmt1 mRNA is undetectable in growth-arrested cells but is induced upon entrance into the S phase of the cell cycle, and until now, the mechanisms responsible for this regulation were unknown. In this report, we demonstrate that the 3'-untranslated region (3'-UTR) of the dnmt1 mRNA can confer a growth-dependent regulation on its own message as well as a heterologous beta-globin mRNA. Our results indicate that a 54-nucleotide highly conserved element within the 3'-UTR is necessary and sufficient to mediate this regulation. Cell-free mRNA decay experiments demonstrate that this element increases mRNA turnover rates and does so to a greater extent in the presence of extracts prepared from arrested cells. A specific RNA-protein complex is formed with the 3'-UTR only in growth-arrested cells, and a UV cross-linking analysis revealed a 40-kDa protein (p40), the binding of which is dramatically increased in growth-arrested cells and is inversely correlated with dnmt1 mRNA levels as cells are induced into the cell cycle. Although ectopic expression of human DNMT1 lacking the 3'-UTR can transform NIH-3T3 cells, inclusion of the 3'-UTR prevents transformation. These results support the hypothesis that deregulated expression of DNMT1 with the cell cycle is important for cellular transformation.

Internal-ribosome-entry-site functional activity of the 3′-untranslated region of the mRNA for the β subunit of mitochondrial H+-ATP synthase

Translation in vitro of the mammalian nucleus-encoded mRNA for the β subunit of mitochondrial H+-ATP synthase (β-mRNA) of oxidative phosphorylation is promoted by a 150 nt translational enhancer sequence in the 3ʹ-untranslated region (3ʹ UTR). Titration of the eukaryotic initiation factor eIF4E with cap analogue revealed that translation of capped β-mRNA was pseudo-cap independent. The 3ʹ UTR of β-mRNA stimulates the translation of heterologous uncapped mRNA species, both when the 3ʹ UTR is placed at the 3ʹ end and at the 5ʹ end of the transcripts. The 3ʹ UTRs of the α subunit of mitochondrial H+-ATP synthase (α-F1-ATPase) and subunit IV of cytochrome c oxidase (COX IV) mRNA species, other nucleus-encoded transcripts of oxidative phosphorylation, do not have the same activity in translation as the 3ʹ UTR of β-mRNA. On dicistronic RNA species, the 3ʹ UTR of β-mRNA, and to a smaller extent that of COX IV mRNA, is able to promote the translation of the second cistron to a level comparable to the activity of internal ribosome entry sites (IRESs) described in picornavirus mRNA species. These results indicate that the 3ʹ UTRs of certain mRNA species of oxidative phosphorylation have IRES-like functional activity. Riboprobes of the active 3ʹ UTRs on dicistronic assays formed specific RNA-protein complexes when cross-linked by UV to proteins of the lysate, suggesting that cytoplasmic translation of the mRNA species bearing an active 3ʹ UTR is assisted by specific RNA-protein interactions.

Internal-ribosome-entry-site functional activity of the 3′-untranslated region of the mRNA for the β subunit of mitochondrial H+-ATP synthase

Translation in vitro of the mammalian nucleus-encoded mRNA for the beta subunit of mitochondrial H(+)-ATP synthase (beta-mRNA) of oxidative phosphorylation is promoted by a 150 nt translational enhancer sequence in the 3'-untranslated region (3' UTR). Titration of the eukaryotic initiation factor eIF4E with cap analogue revealed that translation of capped beta-mRNA was pseudo-cap independent. The 3' UTR of beta-mRNA stimulates the translation of heterologous uncapped mRNA species, both when the 3' UTR is placed at the 3' end and at the 5' end of the transcripts. The 3' UTRs of the alpha subunit of mitochondrial H(+)-ATP synthase (alpha-F1-ATPase) and subunit IV of cytochrome c oxidase (COX IV) mRNA species, other nucleus-encoded transcripts of oxidative phosphorylation, do not have the same activity in translation as the 3' UTR of beta-mRNA. On dicistronic RNA species, the 3' UTR of beta-mRNA, and to a smaller extent that of COX IV mRNA, is able to promote the translation of the second cistron to a level comparable to the activity of internal ribosome entry sites (IRESs) described in picornavirus mRNA species. These results indicate that the 3' UTRs of certain mRNA species of oxidative phosphorylation have IRES-like functional activity. Riboprobes of the active 3' UTRs on dicistronic assays formed specific RNA-protein complexes when cross-linked by UV to proteins of the lysate, suggesting that cytoplasmic translation of the mRNA species bearing an active 3' UTR is assisted by specific RNA-protein interactions.

Identification of a novel cis-element in the 3'-untranslated region of mammalian peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. Growing evidence suggests that the metabolism of PAM messenger RNAs (mRNAs) can be regulated within the cytoplasm. To understand the mechanisms controlling the metabolism of PAM mRNAs, we sought to identify cis elements of the 3'-untranslated region (3'-UTR) of PAM mRNA that are recognized by cytoplasmic factors. From gel retardation assays, one sequence element is shown to form a specific RNA-protein complex. The protein-binding site of the complex was determined by ribonuclease T1 mapping, by blocking the putative binding site with antisense oligonucleotide, and by competition assays. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 20-nucleotide cis element that interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-binding protein complex. The binding activity was redox sensitive. Tissue distribution of the protein in the rat showed a marked tissue-specific expression, with ovary, testis, lung, heart septum, anterior pituitary and hypothalamus containing large amounts compared with liver, ventricle, atrium, and neurointermediate lobe. No binding activity was detectable in pancreas, intestine, or kidney extracts. Northwestern blot analysis of AtT-20 (mouse corticotrope tumor cell line) cytoplasmic extracts revealed a protein of 46 kDa. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 3'-UTR of PAM mRNA from many animal species. Although these data suggest that cis element-binding activity could be a cytoplasmic regulator of PAM mRNA metabolism, the functional consequences of this binding remain to be determined.

Specific binding of a 47-kilodalton protein to the 3' untranslated region of rat transition protein 2 messenger ribonucleic acid.

The synthesis of transition protein (TNP) 2, one of the predominant nuclear proteins of mammalian spermatids, was shown to be posttranscriptionally regulated, by storing the untranslated mRNA for about 3-5 days in the cytoplasm of differentiating spermatids. It has been proposed that binding of a cytoplasmic protein to a conserved motif of 8 nucleotides (nt) in the 3' untranslated region (3'UTR) of TNP2 mRNA is involved in this translational control mechanism. In this report, we show that deletion or variation of the conserved 8-nt motif (GCCAT-CAC) in rat TNP2-3'UTR abolishes the capacity of the in vitro-transcribed RNA to reconstitute specific RNA-protein complexes in RNA bandshift assays. Using Northwestern analysis, we identified specific binding to the TNP2-3'UTR of four proteins of 45, 47, 49, and 60 kDa, all of which are stage-specifically regulated in male germ cell differentiation. Deletion of the 8-nt motif in the 3'UTR specifically prevented binding of the 47-kDa protein, the interaction of which is thought to be mediated by the RNA secondary structure. Analysis of the RNA secondary structure revealed that the 8-nt motif is an essential element of a specific stem-loop structure that is predicted for rat wild-type TNP2-3'UTR. Therefore we assume that the 47-kDa protein plays an important role in specific RNA-protein complex formation of rat TNP2-3'UTR that may be a central event in the translational control of rat TNP2 mRNA.

Cellular Proteins Specifically Bind to the 5′-Noncoding Region of Hepatitis C Virus RNA

Hepatitis C virus (HCV) RNA contains a highly conserved 5′-noncoding region (5′NCR) which may be important in viral multiplication. To study the possible mechanisms of the cellular proteins involved in HCV replication and pathogenesis, a gel mobility shift assay and competition analysis were performed with the HCV 5′NCR. Two specific complexes were formed between the 341-nucleotide RNA of the HCV 5′NCR and proteins of mammalian cell. The specific RNA-protein complexes were maintained in the region of the 5′NCR from nucleotides 131 to 253. Nevertheless, the slower migrating RNA-protein complex failed to form when a polypyrimidine tract sequence (191-UCCUUUCUU-199) in the stem-loop III structure of HCV 5′NCR was changed to 191-UCCUUUggU-199. A uv cross-linking assay further identified two cellular proteins, p87 and p120, that specifically bound to the stem-loop III structure. Mutations at the polypyrimidine tract sequence inhibited the binding of p87, but maintained the ability of the mutant HCV RNA to interact with p120. Translation competition assay demonstrated that the 5′NCR from nt 131 to 253 within the stem-loop III structure is important for the translation of HCV core protein. In addition, p120 and unidentified cellular proteins are likely to be involved in the translation of HCV polyprotein, whereas p87 may play important roles in HCV multiplication other than translation.