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• W2100435038 abstract "Article15 February 1997free access The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein Franck Martin Franck Martin Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author André Schaller André Schaller Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Santa Eglite Santa Eglite Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Daniel Schümperli Daniel Schümperli Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Berndt Müller Corresponding Author Berndt Müller Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Franck Martin Franck Martin Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author André Schaller André Schaller Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Santa Eglite Santa Eglite Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Daniel Schümperli Daniel Schümperli Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Berndt Müller Corresponding Author Berndt Müller Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Search for more papers by this author Author Information Franck Martin1, André Schaller1, Santa Eglite1, Daniel Schümperli1 and Berndt Müller 1 1Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland The EMBO Journal (1997)16:769-778https://doi.org/10.1093/emboj/16.4.769 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The hairpin structure at the 3′ end of animal histone mRNAs controls histone RNA 3′ processing, nucleo-cytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S.cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene. Introduction The replication-dependent animal histone genes (∼50–70 genes in mammals) are transcribed by RNA polymerase II but otherwise follow an expression pathway that is distinct from that of all other protein-coding genes (reviewed in Marzluff and Pandey, 1988; Schümperli, 1988; Osley, 1991). The primary transcripts do not contain introns and are cleaved at their 3′ end in a reaction that is specific for the histone gene family and distinct from cleavage/polyadenylation, resulting in mRNAs without a poly(A) tail. This endonucleolytic cleavage is controlled by two RNA sequence elements: (i) a purine-rich spacer element that serves as an anchoring site for the essential U7 snRNP (Galli et al., 1983; Schaufele et al., 1986; Bond et al., 1991) and (ii) a highly conserved 26 bp sequence encompassing a 6 bp stem–four base loop structure that is important but not essential for maximal processing efficiency (Mowry et al., 1989; Vasserot et al., 1989; Streit et al., 1993). In addition, this hairpin structure, remaining at the 3′ end of the mature histone mRNAs, is involved in further aspects of histone RNA metabolism (reviewed in Marzluff, 1992). It is required for nucleo-cytoplasmic transport (Eckner et al., 1991; Sun et al., 1992), translation (Sun et al., 1992) and stability of histone mRNA (Pandey and Marzluff, 1987). Moreover, it is a key target for a regulated destabilization of histone mRNA occurring upon interruption of cellular DNA replication (Pandey and Marzluff, 1987). Proteins binding to the histone RNA hairpin have been identified in nuclear (Mowry and Steitz, 1987; Vasserot et al., 1989; Pandey et al., 1991; Melin et al., 1992) and polyribosomal extracts (Pandey et al., 1991) and termed hairpin binding (processing) factor (HBF) or stem–loop binding protein (SLBP), respectively. In both cases, a 40–45 kDa protein was cross-linked to hairpin RNA by UV irradiation (Pandey et al., 1991), and recent experiments indicate that the polysomal protein can complement HBF-depleted nuclear extracts in histone RNA 3′ processing, suggesting that the two proteins are identical or share at least one common polypeptide (Dominski et al., 1995). Attempts at purification have provided enriched fractions which were useful for further biochemical analysis (Dominski et al., 1995; Hanson et al., 1996; A.Schaller, F.Martin and B.Müller, in preparation) but have not yet yielded the protein(s) in sufficient quantity for direct amino acid sequence determination. Using the newly developed Saccharomyces cerevisiae three-hybrid system for selection/screening of RNA binding proteins (Sengupta et al., 1996), we have now isolated a human cDNA clone for a protein that binds specifically to the histone RNA hairpin. We demonstrate that this protein participates in histone pre-mRNA 3′ end processing. To indicate that we have cloned the RNA binding component of HBF/SLBP, we will henceforth refer to this protein/gene as HBP for (histone) hairpin binding protein. We also describe the cDNA and genomic structure as well as the expression pattern in different human tissues. Except for a putative Caenorhabditis elegans homologue, HBP has no significant homology to any other proteins or motifs in the SWISSPROT and PROSITE databases, and may thus represent a new type of RNA binding protein. Results Cloning of HBP cDNA The yeast three-hybrid system (Sengupta et al., 1996) has been derived from the two-hybrid system commonly used for studying protein–protein interactions (Fields and Song, 1989; Durfee et al., 1993). It allows for a selection for specific RNA binding proteins using the following features: two reporter genes (HIS3 and lacZ, both preceded by a DNA binding site for LexA protein) and a fusion between lexA and the gene for phage MS2 coat protein are all stably integrated into the genome of S.cerevisiae strain L40-coat. Thus the LexA–MS2 coat fusion protein can bind to the LexA sites but this does not activate the reporter genes because of lack of an activation domain able to attract and stimulate the transcription machinery (Figure 1A). The second hybrid is an RNA, transcribed in vivo from a plasmid, that contains two binding sites for MS2 coat protein and a site for the RNA binding protein of interest (Figure 1B). The third hybrid should be a fusion protein between the corresponding RNA binding protein and the Gal4 activation domain. This can be provided on an appropriate plasmid expression vector or selected from a cDNA library cloned in such a vector. Figure 1.Introduction to assay systems. (A) The yeast three-hybrid system was developed by M.Wickens, S.Fields and co-workers (Sengupta et al., 1996) and depends on the activation of reporter genes (HIS3 and lacZ) by an RNA-mediated interaction between the LexA–MS2 coat fusion protein and the Gal4 cDNA fusion protein. The structure of the RNA molecules used is as shown in (B). Reporter genes as well as the gene for the LexA–MS2 coat fusion protein are integrated in the genome of S.cerevisiae strain L40-coat. (B) Structure of RNA sequences used in the three-hybrid selection. RNAs containing either wild-type histone hairpin sequences or mutant hairpin sequences (marked HP) 3′ of the RNase P leader sequence and 5′ of two serial MS2 RNA elements were encoded by plasmids pIII/wtHP-MS2 and pIII/mutHP-MS2. The hairpin sequences are identical to wtHP and mutHP RNAs shown in (C). (C) RNA molecules used to detect the HBP by EMSA. Download figure Download PowerPoint To perform the three-hybrid selection, we introduced the wtHP sequence into the pIII/MS2-2 plasmid to yield plasmid pIII/wtHP-MS2 encoding a hybrid RNA containing the wild-type histone hairpin structure (Figure 1B and C). After this plasmid was introduced into S.cerevisiae strain L40-coat by URA3 selection, we transformed the resulting strain with a Gal4 activation domain-tagged cDNA library from human lymphocytes (Durfee et al., 1993). Titration on plates lacking uracil and leucine showed that the library was producing 300 000 original transformants. The culture was plated out on artificial medium lacking uracil, leucine and histidine to select for the presence of the plasmids and activation of the HIS3 reporter gene. After 5 days of selection, four His+ S.cerevisiae colonies were observed. The four colonies (clones 1–4) were tested on URA-LEU-HIS selective media containing 5-bromo-4-chloro-3-indolyl-β-D-galactosidase, to visualize expression of the lacZ reporter gene. All four grew to blue colonies, indicating that the lacZ gene was also activated (Table I). We then isolated derivatives which had lost either the URA3-containing pIII/wtHP-MS2 plasmid or the LEU2-containing cDNA plasmid. For all clones, the His+ and lacZ+ phenotypes were lost with the Gal4 cDNA expression plasmid. However, only clone 2 also lost both phenotypes when the pIII/wtHP-MS2 plasmid was removed. The proteins encoded by the other three clones (1, 3 and 4) which retained both phenotypes therefore appeared to activate the reporter genes independently of the hybrid histone hairpin RNA, e.g. by binding either to the promoter DNA or to the LexA–MS2 coat fusion protein. Table 1. His+ and lacZ+ phenotypes of clone 2 are wtHP RNA-mediated and dependent on cDNA Clone 1 2 3 4 Phenotype His/lacZ His/lacZ His/lacZ His/lacZ Original transformants +/+ +/+ +/+ +/+ after loss of pIII/wtHP-MS2a +/+ −/− +/+ +/+ after loss of pACT-cDNA −/− −/− −/− −/− pACT-cDNA plasmids retransformed into yeast L40-coat: without pIII plasmid +/+ −/− n.d. +/+ with pIII/IRE-MS2 plasmidb +/+ −/− n.d. +/+ with pIII/wtHP-MS2 +/+ +/+ n.d. +/+ with pIII/mutHP-MS2c +/+ −/− n.d. +/+ a For the structure of pIII/wtHP-MS2 RNA, see Figure 1B and C b Containing iron response element RNA instead of wtHP RNA. c Containing mutHP RNA (Figure 1C) instead of wtHP RNA. Three of the four cDNA plasmids (clones 1, 2 and 4) were recovered, amplified in Escherichia coli and then re-transformed into a series of S.cerevisiae L40-coat strains containing either no or different variants of the pIII RNA plasmid (Table I). In plasmid pIII/mutHP-MS2, the wtHP sequence was replaced by the mutant mutHP sequence (RNA structure and sequences are shown in Figure 1B and C) and plasmid pIII/IRE-MS2 contained an iron response element instead of the wtHP sequence (Sengupta et al., 1996). Clones 1 and 4 produced His+ and lacZ+ phenotypes irrespective of whether the cell contained no pIII plasmid or plasmids pIII/IRE-MS2, pIII/wtHP-MS2 or pIII/mutHP-MS2, confirming that they activated both reporter genes by an RNA-independent mechanism. However, clone 2, the candidate for HBP cDNA, produced His+ and lacZ+ phenotypes only in the S.cerevisiae strain harbouring the pIII/wtHP-MS2 plasmid. We then tested whether extracts prepared from the original S.cerevisiae transformants produce an RNA binding activity with the same characteristics as mammalian HBP. After incubation with radiolabelled 34 nucleotide wtHP RNA (Figure 1C) and native gel electrophoresis (electrophoretic mobility shift assay, EMSA), only clone 2 produced a ribonucleoprotein (RNP) complex with a strongly reduced mobility (Figure 2A, lane 4), whereas clones 1, 3 and 4 only produced faint, faster migrating and presumably unspecific complexes (lanes 3, 5 and 6). The complex produced by S.cerevisiae clone 2 had a slower mobility than the murine HBP complex (compare lanes 2 and 4), consistent with the fact that the fusion protein additionally contained the Gal4 activation domain. We further compared the RNA binding specificity of the Gal4–HBP fusion protein with a highly enriched HBP fraction prepared from calf thymus using ion exchange chromatography (CT fraction V; A.Schaller, F.Martin and B.Müller, in preparation). Binding reactions were performed in the absence or presence of varying amounts of unlabelled wtHP, mutHP or cgHP competitor RNAs (sequences are shown in Figure 1C). Whereas mutHP contained a completely unrelated 6 bp stem–four base loop structure, in cgHP, only the two lowest base pairs of the stem were inverted from G–C to C–G. This mutant was reported to have ∼3% of the wild-type affinity for HBP (Pandey et al., 1994; Williams and Marzluff, 1995). The HBP-specific complexes produced by CT fraction V and by the extract from S.cerevisiae clone 2 were both partly competed by a 10-fold excess (Figure 2B, lanes 2 and 9, respectively) and inhibited by a 100-fold excess of wtHP RNA (lanes 3 and 10). In contrast, neither of the two mutant RNAs were able to inhibit the formation of HBP-specific complexes at these concentrations (lanes 4–7 and 11–14). The same RNA binding activity was also detected after re-transformation of the cDNA plasmid from clone 2 into S.cerevisiae strain L40-coat (data not shown), indicating that clone 2 cDNA encodes a protein with RNA binding specificity identical to mammalian HBP. Figure 2.cDNA from clone 2 encodes a Gal4–HBP fusion protein. (A) Extracts prepared from the four selected transformants were tested for the presence of HBP. Lane 1, 32P-labelled wtHP RNA. Lane 2, 12 μl of mouse K21 cell nuclear extract (6 mg/ml) was mixed with 32P-labelled wtHP RNA and incubated in 20 μl as described in Materials and methods. Lanes 3–6, as lane 2, but the nuclear extract was replaced by extracts prepared from the transformants 1–4 (3, 1.6, 1.7 and 2.2 mg/ml protein, respectively). Reaction products were analysed by EMSA as described in Materials and methods. (B) Comparison of RNA binding specificity of HBP encoded by clone 2 and of HBP enriched from calf thymus. Lanes 1–7, 25 fmol of 32P-labelled wtHP RNA were incubated with 5 μl of extract prepared from clone 2 in a final volume of 10 μl as described in Materials and methods. Lanes 8–14, as lanes 1–7, but yeast extract was replaced by calf thymus fraction V (40 μg/ml). Competitor RNAs were included as follows: lanes 2 and 9, 250 fmol of wtHP RNA; lanes 3 and 10, 2.5 pmol of wtHP RNA; lanes 4 and 11, 250 fmol of mutHP RNA; lanes 5 and 12, 2.5 pmol of mutHP RNA; lanes 6 and 13, 250 fmol of cgHP RNA; lanes 7 and 14, 2.5 pmol of cgHP RNA. Download figure Download PowerPoint The sequence of HBP The cDNA insert from clone 2 was subcloned into a pBluescript vector and its nucleotide sequence determined (EMBL nucleotide sequence database accession No. Z71188). Inspection of the sequence revealed a cDNA insert of 1716 bp with an additional tail of 22 A residues. The poly(A) tail is preceded by a perfect AATAAA sequence ending 17 nucleotides before the poly(A) addition site. The cDNA insert contains an open reading frame (ORF) beginning at the very 5′ end (reading frame 3) and ending with a TAA stop codon at position 915. The first ATG at position 105 (Figure 3A) is in a favourable sequence context for an initiation codon (Kozak, 1986). The ORF, from this position onwards, encodes a protein of 270 amino acids with a calculated Mr of 31 186 Da. This is lower than the 40–45 kDa estimated by SDS–PAGE of the murine protein labelled by UV cross-linking to radiolabelled RNA (Pandey et al., 1991; A.Schaller and B.Müller, unpublished observation). However, we have expressed an HBP cDNA fragment starting from the ATG at position 105 in S.cerevisiae using a galactose-inducible expression system (see Materials and methods). The molecular mass of this protein determined by UV cross-linking was ∼41 kDa (data not shown). In addition, we have translated the cDNA in a wheat germ extract (the AUG at position 105 being the first possible initiation codon), yielding a protein with an apparent Mr of 43 kDa (see below). These observations strongly suggest that we have isolated a virtually complete cDNA and that translation starts at the AUG at position 105. Figure 3.(A) Promotor region and first exon of the HBP gene. The sequence of the HBP cDNA was deposited in the EMBL nucleotide sequence database (accession No. Z71188). Genomic DNA sequences are from McCombie and colleagues (McCombie et al. 1992; DDBJ/EMBL/GenBank nucleotide sequence database accession No.M63480). The TATA box-like sequence and possible SP1 recognition sites are underlined. The translated part of exon 1 is boxed; +1 marks the beginning of the cDNA. (B) Deduced amino acid sequence alignment between human HBP and the putative C.elegans histone HBP Yrm1 (SWISSPROT database accession No. Q09599) as determined using the BlastP program (Altschul et al., 1990). Only the Yrm1 region with highest homology to HBP is shown. Identical amino acids (1, 53%) and conserved amino acids (:, 12 %) are indicated. Download figure Download PowerPoint Nucleotide sequence comparisons revealed that the genomic region of the HBP gene had been sequenced previously (McCombie et al., 1992). In this report, three human cosmids, located within what was then the candidate region for the Huntington's disease gene (chromosome 4p16.3), were sequenced, yielding a contig of 58 864 bp. Based on the location of CpG-rich islands and ORFs, three genes were predicted to lie in this region, one of which was characterized further by cDNA cloning and named hda1-1. The 5′ end of the HBP cDNA is located in the next CpG island downstream of the hda1-1 gene. If the long ORF of HBP cDNA is prolonged into the 5′-flanking genomic region, it meets a stop codon 19–17 nucleotides upstream of the cDNA's 5′ end (Figure 3A). These 19 bp contain neither an additional in-frame ATG nor a sequence resembling a 3′ splice site. Therefore, the first ATG and hence the correct initiation codon is the one at position 105. Furthermore, 55–50 nucleotides upstream from the cDNA's 5′ end is a TATA box-like sequence, CATAAA, flanked by two recognition sites for transcription factor SP1. By comparison with the genomic sequence, the HBP gene comprises eight exons of ≥163, 117, 105, 60, 138, 150, 67 and 916 bp, interrupted by introns of 139, 8133, 3524, 309, 3206, 1361 and 1060 bp, respectively. This adds up to ≥19 448 bp for the entire gene. Comparison of the amino acid sequence predicted by the ORF from position 105–915 with the SWISSPROT library revealed only one related protein, a putative 41.5 kDa protein predicted from the C.elegans genome project (SWISSPROT databank accession No. Q09599), but no known protein with significant homologies. This protein displayed a significant degree of sequence similarity to residues 33–195 of human HBP, but the highest conservation was observed for residues 130–195 (Figure 3B). Cloning and in vitro translation of the corresponding C.elegans cDNA produced a protein of apparent Mr ∼58 kDa which bound specifically to RNA containing a C.elegans histone hairpin (data not shown; a detailed comparison of the human and C.elegans proteins will be presented elsewhere). Further analysis of the PROSITE library revealed no known motifs besides a potential nuclear localization signal RKRR (amino acids 31–34) and a number of putative phosphorylation sites. The HBP gene product binds histone RNA hairpin structures To exclude that the Gal4 activation domain was involved in RNA binding, the HBP cDNA by itself was transcribed and translated in vitro in wheat germ extract. The product was radiolabelled by the inclusion of [35S]methionine and analysed by SDS–PAGE. Figure 4A shows that the main translation product, a 43 kDa protein, and some minor smaller peptides were formed only when the cDNA was in the sense orientation (lane 2). Reactions with the ‘antisense’ cDNA did not lead to any protein synthesis (lane 1). Figure 4.Translation of HBP cDNA in vitro. (A) Analysis of translation products. pBluescript-HBP (antisense cDNA orientation) or pBluescript-rHBP DNA (sense cDNA orientation) were transcribed and translated in wheat germ extract as described in Materials and methods. Five μl of each reaction were precipitated by addition of trichloroacetic acid, analysed by SDS–10% polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the pre-stained marker proteins are indicated. (B) HBP translated in vitro binds histone hairpin RNA. Ten μl of K21 nuclear extract (lane 1) or 10 μl of translation reaction mixtures containing pBluescript-HBP (antisense cDNA orientation; lane 2) or pBluescript-rHBP DNA (sense cDNA orientation; lanes 3–5) were mixed with radiolabelled wtHP RNA and the reaction products analysed by EMSA as described in Materials and methods. The reactions in lane 4 and 5 were supplemented with 2.5 pmol of either wtHP or mutHP competitor RNA. Download figure Download PowerPoint To confirm that the 43 kDa protein contained the RNA binding activity, the translation mixtures were tested directly in an RNA binding assay. Binding reactions with the extract containing the 43 kDa protein and wtHP RNA formed an RNP complex which co-migrated with the one formed by the K21 nuclear extract (Figure 4B lanes 3 and 1, respectively). An additional, faster migrating complex, presumably originating from one of the minor translation products, was also formed in these reactions. Both complexes were absent in incubations with the translation reaction of ‘antisense’ cDNA (lane 2) and were sensitive to the presence of wtHP but not mutHP competitor RNA (lanes 4 and 5, respectively). This confirmed that the cDNA-encoded protein has the binding specificity expected of mammalian HBP as well as the correct electrophoretic mobility both on SDS–polyacrylamide and native gels. The HBP participates in histone pre-mRNA processing To test whether the recombinant HBP was functional in histone pre-mRNA 3′ end processing, we used two different sources of K21 mouse cell nuclear extract deficient in HBP. Fractionation of K21 extract by MonoQ column chromatography separated hairpin binding factor (HBF)/HBP from U7 snRNP (Vasserot et al., 1989), leading to side fractions with low processing activity. In our preparation, fraction 20 was enriched for HBF but lacked U7 snRNP, while fraction 24 was enriched for U7 snRNP and contained little HBF (A.Schaller, F.Martin and B.Müller, in preparation). Incubation of a histone H4 RNA 3′ end fragment encompassing the processing site (Vasserot et al., 1989) with fraction 24 led to 1.7% of the RNA being processed (Figure 5A, lanes 2, 5 and 10; quantitation of two separate experiments is shown in Figure 5B), while ∼58% was processed in a reaction with unfractionated nuclear extract from K21 cells (lane 1). In incubations with fraction 20, radiolabel at the position of the processing product was at background level (0.7%; lane 3). Mixing fraction 20 with fraction 24 increased cleavage of the histone RNA ∼2.6-fold (lane 4), illustrating that the addition of HBF stimulated the activity of U7 snRNPs in this in vitro assay. However, the level of processing did not reach the level obtained with unfractionated K21 extract, indicating that, in addition to HBF, other factor(s) may be missing. Figure 5.Complementation of histone RNA 3′ processing using nuclear extract depleted of HBP by MonoQ column chromatography and S.cerevisiae extract containing recombinant HBP. (A) Processing reactions with a 32P-labelled 69 nucleotide H4 RNA fragment contained mouse K21 cell nuclear extract fractionated by MonoQ column chromatography and were performed and analysed by denaturing gel electrophoresis as described in Materials and methods. Reactions contained 5 μl of K21 nuclear extract (5 mg/ml) (U7 snRNP and HBP; lane 1), 2.5 μl of MonoQ fraction 24 (1 mg/ml) (enriched for U7 snRNP; lanes 2, 5 and 10), 2.5 μl of MonoQ fraction 20 (0.8 mg/ml) (enriched for HBP/HBF; lane 3) or 2.5 μl of fraction 20 mixed with 2.5 μl of fraction 24 (lane 4). Reactions in lanes 7–9 contained 2.5 μl of MonoQ fraction 24 and 2.5 μl of BJ5465/pFMM5 extract (4 mg/ml) containing HBP. Reactions in lanes 12–14 contained 2.5 μl of MonoQ fraction 24 and 2.5 μl of BJ5465 extract (4 mg/ml). Lanes 8 and 13 additionally contained 2.5 pmol of unlabelled wtHP competitor RNA. Lanes 9 and 14 aditionally contained 2.5 pmol of unlabelled mutHP competitor RNA. The reaction in lane 6 was with 2.5 μl of BJ5465/pFMM5 extract only and the reaction in lane 11 with 2.5 μl of BJ5465 extract only. Molecular weight marker M is pBR322 DNA cleaved with HpaII. (B) Quantitation of two independent experiments using a phosphorimager as described in Materials and methods. Processing reactions contained as indicated: K21, K21 nuclear extract; MQ20, MonoQ fraction 20; MQ24, MonoQ fraction 24; ScHBP, BJ5465/pFMM5 extract; Sc, BJ5465 extract; wtHP, wtHP competitor RNA; mutHP, mutHP competitor RNA. Results from the experiment in (A) are shown as black bars. For simplicity, only the mean (1.7%) of the three incubations with MonoQ fraction 24 in (A), lanes 2, 5 and 10 is shown (lane 2, 1.7%; lane 5, 1.8%; lane 10, 1.6%). Download figure Download PowerPoint Using this complementation assay, we detected a stimulation of processing of fraction 24 by wheat germ extract containing recombinant HBP (data not shown) and by extract prepared from a S.cerevisiae strain expressing recombinant HBP from the ATG at position 105 (see Materials and methods). Addition of extract prepared from S.cerevisiae expressing HBP stimulated processing 2.6-fold (Figure 5A, lane 7), whereas no significant increase was observed with control yeast extract (lane 12), indicating that the presence of recombinant HBP in the extract contributed to processing. This was confirmed by the observation that inclusion of wtHP competitor RNA (lane 8), but not mutHP competitor RNA (lane 9), reduced processing to the level of processing obtained with fraction 24 alone. In contrast, product formation in the presence of control extract was not affected by the addition of either kind of competitor RNA (lanes 13 and 14). As expected, neither of the two yeast extracts showed any processing activity on its own (lanes 6 and 11). In another series of experiments, we depleted K21 extract of HBP using biotinylated wtHP RNA and streptavidin–agarose (A.Schaller and B.Müller, unpublished results). This led to an ∼6-fold reduction in processing activity (Figure 6A, lanes 1 and 2; quantitated in Figure 6B), and mixing of HBP-depleted with untreated extract showed that the depleted extract did not inhibit processing (lane 3). A 3.7-fold stimulation of processing could be achieved using the HBF-containing MonoQ fraction 20 (lane 14) already used in the above experiment. A 3-fold stimulation was obtained with an enriched preparation of bovine HBP (lane 10) and, more importantly, a 2-fold stimulation was also achieved with an identically fractionated preparation of recombinant HBP from cDNA-expressing S.cerevesiae strain BJ5465/pFMM5 (lane 6). Very similar effects were obtained in three separate experiments (Figure 6B) and in additional experiments which are not shown. With all three sources of HBP, this stimulation was prevented by inclusion of wtHP (Figure 6A, lanes 7, 11 and 15), but not mutHP competitor RNA (lanes 8, 12 and 16). These experiments demonstrate that recombinant HBP produced in S.cerevisiae is functional in histone pre-mRNA processing. Figure 6.Complementation of histone RNA 3′ processing in nuclear extract depleted of HBP using biotinylated histone hairpin RNA with enriched recombinant HBP. (A) Processing reactions were performed and analysed by denaturing gel electrophoresis as described in Materials and methods. Reactions contained 2.5 μl of K21 nuclear extract (3.1 mg/ml) (lane 1) or 2.5 μl of nuclear extract depleted of HBP with biotinylated histone hairpin RNA as described in Materials and methods (1.8 mg/ml) (lanes 2–4, 6–8, 10–12 and 14–16). Incubations of reactions in lanes 2 and 4 were without further addition." @default.
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• W2100435038 date "1997-02-15" @default.
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• W2100435038 title "The gene for histone RNA hairpin binding protein is located on human chromosome 4and encodes a novel type of RNA binding protein" @default.
• W2100435038 cites W1570594834 @default.
• W2100435038 cites W1589290041 @default.
• W2100435038 cites W1831176116 @default.
• W2100435038 cites W1966694632 @default.
• W2100435038 cites W1977186059 @default.
• W2100435038 cites W1979060215 @default.
• W2100435038 cites W1993050840 @default.
• W2100435038 cites W1995562634 @default.
• W2100435038 cites W2007720824 @default.
• W2100435038 cites W2012565545 @default.
• W2100435038 cites W2017541340 @default.
• W2100435038 cites W2020964929 @default.
• W2100435038 cites W2025347522 @default.
• W2100435038 cites W2027300279 @default.
• W2100435038 cites W2029953457 @default.
• W2100435038 cites W2046134908 @default.
• W2100435038 cites W2052108496 @default.
• W2100435038 cites W2053187594 @default.
• W2100435038 cites W2054331233 @default.
• W2100435038 cites W2055043387 @default.
• W2100435038 cites W2072728868 @default.
• W2100435038 cites W2085272777 @default.
• W2100435038 cites W2086660631 @default.
• W2100435038 cites W2108053853 @default.
• W2100435038 cites W2120306478 @default.
• W2100435038 cites W2120357602 @default.
• W2100435038 cites W2131481178 @default.
• W2100435038 cites W2134088228 @default.
• W2100435038 cites W2138270253 @default.
• W2100435038 cites W2139159103 @default.
• W2100435038 cites W2142946131 @default.
• W2100435038 cites W2154128645 @default.
• W2100435038 cites W2295015362 @default.
• W2100435038 cites W2401820952 @default.
• W2100435038 cites W296057586 @default.
• W2100435038 cites W4242773288 @default.
• W2100435038 cites W9166349 @default.
• W2100435038 cites W9501228 @default.
• W2100435038 doi "https://doi.org/10.1093/emboj/16.4.769" @default.
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