PIPPin Is a Brain-specific Protein That Contains a Cold-shock Domain and Binds Specifically to H1° and H3.3 mRNAs

During maturation of mammalian brain, variants of both linker (i.e. H1°) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a cold-shock domain, binds with high specificity to the transcripts that encode H1° and H3.3 histone variants. Both mRNAs are bound through the very end of their 3′-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation. During maturation of mammalian brain, variants of both linker (i.e. H1°) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a cold-shock domain, binds with high specificity to the transcripts that encode H1° and H3.3 histone variants. Both mRNAs are bound through the very end of their 3′-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation. cold shock domain untranslated region nucleotide(s) maltose-binding protein phosphate-buffered saline polyacrylamide gel electrophoresis microtubule-associated protein During development of an organism and tissue differentiation, chromatin must be remodeled to permit entrance of transcription factors and hence expression of genes at the right places and times. Although a critical moment for setting new patterns of chromatin organization is the S phase of the cell cycle, it is now clear that chromatin can be remodeled also in the absence of DNA replication, by energy consuming complexes (1Griffin Burns L. Peterson C.L. Biochim. Biophys. Acta. 1997; 1350: 159-168Crossref PubMed Scopus (32) Google Scholar, 2Ito T. Bulger M. Pazin M.J. Kobayashi R. Kadonaga J.T. Cell. 1997; 90: 145-155Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 3Varga-Weisz P.D. Wilm M. Bonte E. Dumas K. Mann M. Becker P.B. Nature. 1997; 388: 598-602Crossref PubMed Scopus (436) Google Scholar, 4Utley R.T. Ikeda K. Grant P.A. Côté J. Steger D.J. Eberharter A. John S. Workman J.L. Nature. 1998; 394: 498-502Crossref PubMed Scopus (447) Google Scholar). The possibility that remodeling also allows entrance, at topologically defined regions of the nucleus, of specific histone isotypes, which might locally modify chromatin organization even more, is provocative and deserves of attention. We previously demonstrated that, in the developing rat brain, the concentration of H1° and H3.3 mRNAs decreases between the embryonal day 18 (E18) and the postnatal day 10 (P10), whereas the corresponding genes are transcribed at the same rate at any stage studied, suggesting that the two genes are regulated mainly at post-transcriptional level (5Castiglia D. Cestelli A. Scaturro M. Nastasi T. Di Liegro I. Neurochem. Res. 1994; 19: 1531-1537Crossref PubMed Scopus (31) Google Scholar, 6Scaturro M. Cestelli A. Castiglia D. Nastasi T. Di Liegro I. Neurochem. Res. 1995; 20: 969-976Crossref PubMed Scopus (27) Google Scholar). As post-transcriptional control processes, including regulation of splicing (7Matsumoto K. Wassarman K.M. Wolffe A.P. EMBO J. 1998; 17: 2107-2121Crossref PubMed Scopus (177) Google Scholar), vectorial transport of mature mRNAs (8St Johnston D. Cell. 1995; 81: 161-180Abstract Full Text PDF PubMed Scopus (510) Google Scholar, 9Lee M.S. Silver P.A. Curr. Opin. Genet. Dev. 1997; 7: 212-219Crossref PubMed Scopus (32) Google Scholar, 10Oleynikov Y. Singer R.H. Trends Cell Biol. 1998; 8: 381-383Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), regulation of mRNA stability (11Nielsen D.A. Shapiro D.J. Mol. Endocrinol. 1990; 4: 953-957Crossref PubMed Scopus (136) Google Scholar, 12Beelman C.A. Parker R. Cell. 1995; 81: 179-183Abstract Full Text PDF PubMed Scopus (573) Google Scholar, 13Decker C.J. Parker R. Trends Biochem. Sci. 1994; 19: 336-340Abstract Full Text PDF PubMed Scopus (212) Google Scholar), and availability for translation (14Jackson R.J. Cell. 1993; 74: 9-14Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 15Hentze M.W. Curr. Opin. Cell Biol. 1995; 7: 393-398Crossref PubMed Scopus (64) Google Scholar), are mediated by several classes of RNA-binding proteins (for review, see Refs. 16Burd C.G. Dreyfuss G. Science. 1994; 265: 615-662Crossref PubMed Scopus (1734) Google Scholar, 17Siomi H. Dreyfuss G. Curr. Opin. Genet. Dev. 1997; 7: 345-353Crossref PubMed Scopus (232) Google Scholar, 18Weeks K.M. Curr. Opin. Struct. Biol. 1997; 7: 336-342Crossref PubMed Scopus (110) Google Scholar), it is likely that developing rat brain contains mRNA-binding factors involved in the binding and regulation of mRNAs encoding histone variants. We reported in a previous paper (19Castiglia D. Scaturro M. Nastasi T. Cestelli A. Di Liegro I. Biochem. Biophys. Res. Commun. 1996; 218: 390-394Crossref PubMed Scopus (29) Google Scholar) cloning and analysis of a cDNA encoding a putative RNA-binding protein, specifically expressed in the rat brain and conserved from Drosophila melanogaster to man. The protein, that contains two regions with chemical homology to double-stranded RNA-binding motifs (16Burd C.G. Dreyfuss G. Science. 1994; 265: 615-662Crossref PubMed Scopus (1734) Google Scholar) was called PIPPin after the first four amino acids of the second of these motifs (PIPP, in one-letter code). Here we report that PIPPin contains also a potential cold-shock domain (CSD1; for review, see Refs.20Graumann P.L. Marahiel M.A. Trends Biochem. Sci. 1998; 23: 286-290Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar and 21Matsumoto K. Wolffe A.P. Trends Cell Biol. 1998; 8: 318-323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Within the latter, two short sequences are particularly interesting as they correspond to the so called ribonucleoprotein motifs 1 and 2 (RNP1 and RNP2), respectively, and are conserved among PIPPin and several other prokaryotic as well as eukaryotic nucleic acid-binding proteins. The presence, in PIPPin, of a CSD flanked on both sides by putative double-stranded RNA-binding motifs strongly suggested that the protein could really be an RNA-binding factor. The results reported in the present paper clearly demonstrate that this is in fact the case. Moreover, we found that PIPPin binds preferentially to RNAs encoding H3.3 and H1° histone variants. Using an in vitro culture system, we further demonstrate that PIPPin is present both in the cytoplasm and nucleus of nerve cells; however, its ability to bind RNA seems to be confined to the nucleus. We report that about one-half of nuclear PIPPin is more acidic (pI ≅ 6.0) than expected (pI = 7.7), thus suggesting that the protein would be post-translationally modified (perhaps by phosphorylation), in order to bind RNA. Harlan Sprague-Dawley rats and New Zealand rabbits (Stefano Morini, San Polo d'Enza, Italy) were housed and handled according to European Community Council Directive 86/609, OJL 358 1, 12 December 1987 (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985). Neuronal cultures were prepared from embryonic day 16 rat cerebral cortices, and cultured for 5–8 days in a selective, serum-free medium, on Primaria tissue culture dishes (Falcon), precoated with laminin (Roche Molecular Biochemicals), as described in detail elsewhere (22Cestelli A. Savettieri G. Ferraro D. Vitale F. Dev. Brain Res. 1985; 22: 219-227Crossref Scopus (36) Google Scholar, 23Savettieri G. Mazzola G.A. Rodriguez Sanchez M.B. Caruso G. Di Liegro I. Cestelli A. Cell. Mol. Neurobiol. 1998; 18: 369-378Crossref PubMed Scopus (13) Google Scholar). To obtain total post-nuclear, mitochondrial, microsomal, and post-microsomal (S-100) cell subfractions, fresh tissues from developing and adult rats or cultured neurons were processed as described previously (24Scaturro M. Nastasi T. Raimondi L. Bellafiore M. Cestelli A. Di Liegro I. J. Biol. Chem. 1998; 273: 22788-22791Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Nuclear extracts were prepared according to Dignam et al. (25Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Protein concentration of all fractions was determined by the method described by Lowry et al. (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), using bovine serum albumin as a standard. Some of the plasmids used for in vitro transcription of both radiolabeled and cold RNA probes were already described elsewhere (24Scaturro M. Nastasi T. Raimondi L. Bellafiore M. Cestelli A. Di Liegro I. J. Biol. Chem. 1998; 273: 22788-22791Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The other plasmids were constructed ad hoc, by subcloning different portions of the original inserts into the Bluescript KS+ plasmid (Stratagene). Briefly, to synthesize transcripts corresponding to most of the H1° mRNA, we used as template the original pMH1° (EMBL accession number X70685; Ref. 27Castiglia D. Gristina R. Scaturro M. Di Liegro I. Nucleic Acids Res. 1993; 21: 1674Crossref PubMed Scopus (25) Google Scholar), that contains an insert of 1711 nucleotides (nt). To obtain smaller H1° transcripts, we used the subclones described previously (24Scaturro M. Nastasi T. Raimondi L. Bellafiore M. Cestelli A. Di Liegro I. J. Biol. Chem. 1998; 273: 22788-22791Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) and reported in Fig. 3 B (H1°, a to d). To synthesize H3.3 mRNA, we used as template pDH3, obtained by ligation of the 5′ region of PDH 33-2- and the 3′-region of pDH 33-1 inserts (EMBL accession numberX73683; Ref. 5Castiglia D. Cestelli A. Scaturro M. Nastasi T. Di Liegro I. Neurochem. Res. 1994; 19: 1531-1537Crossref PubMed Scopus (31) Google Scholar). To obtain smaller transcripts, corresponding to different portions of the untranslated region 3′ to the coding portion (3′-UTR) of H3.3 mRNA, we subcloned the following two regions of the H3.3 insert: 1) from nt 537 to nt 1107 (pR4: corresponding to the whole 3′-UTR, indicated as R4, in Fig. 3 B); 2) from nt 909 to 1107 (pM4: corresponding to the last 198 nt of 3′-UTR, indicated as M4, in Fig. 3 B). To synthesize the c-erbA α2 transcript, we used the pA1.3K plasmid, that contains an insert of about 1200 nt, corresponding to the full-length c-erbA α2 mRNA (28Castiglia D. Cestelli A. Di Liegro C. Bonfanti L. Di Liegro I. Cell. Mol. Neurobiol. 1992; 12: 259-272Crossref PubMed Scopus (10) Google Scholar). Finally, to synthesize a maltose-binding protein (MBP-)/PIPPin fusion protein, the coding region of the PIPPin insert, from the Cx1 plasmid (accession number X89962: Ref. 19Castiglia D. Scaturro M. Nastasi T. Cestelli A. Di Liegro I. Biochem. Biophys. Res. Commun. 1996; 218: 390-394Crossref PubMed Scopus (29) Google Scholar) was amplified, by polymerase chain reaction, using the following primers: 5′-dAGCGAATTCATGACATCAGAGTCTACATGACCC-3′; The and and respectively, to cloning of the into the plasmid the subclones were from both by the radiolabeled of proteins were by D.J. J. Mol. Biol. 1990; PubMed Scopus Google Scholar) at the for and using The corresponding accession for and E. E. D. melanogaster the amino of PIPPin was to The at C. J. Mol. Biol. 1993; PubMed Scopus Google Scholar, C. 1994; 19: PubMed Scopus Google Scholar) and a domain was F. J. D. Nucleic Acids Res. 1998; PubMed Scopus Google Scholar). by was also used to the sequences of H1° and H3.3 The fusion protein was expressed in E. and from by on New Zealand rabbits were with of fusion protein, in were with of protein, in The first was 2 after the first and more were at the and were and at and fractions were obtained from total serum by Briefly, the fusion protein were first on the fusion protein, bound to and from the with the second were from the obtained in the first by on The bound by the is enriched in bound were with the fractions were and at RNA from cultured neurons was according to and Biochem. PubMed Scopus Google Scholar), and by electrophoresis on RNA was to and to PIPPin as already described (19Castiglia D. Scaturro M. Nastasi T. Cestelli A. Di Liegro I. Biochem. Biophys. Res. Commun. 1996; 218: 390-394Crossref PubMed Scopus (29) Google Scholar). of total or cell were by electrophoresis on polyacrylamide and as described elsewhere G. Mazzola G.A. Rodriguez Sanchez M.B. Caruso G. Di Liegro I. Cestelli A. Cell. Mol. Neurobiol. 1998; 18: 369-378Crossref PubMed Scopus (13) Google Scholar). electrophoretic analysis was as described by et al. D. G. J. 1993; Google Scholar), with in the in the first and in the second The of was determined by using proteins of as were by with which the to the and used as for in vitro transcription of and transcripts, from the and or and RNA polymerase according to were as described elsewhere (24Scaturro M. Nastasi T. Raimondi L. Bellafiore M. Cestelli A. Di Liegro I. J. Biol. Chem. 1998; 273: 22788-22791Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To any H3.3 RNA binding as PIPPin, in or nuclear cell fractions to 1 of total were with of H3.3 RNA and to The putative PIPPin-RNA complexes were from the with To putative PIPPin-RNA obtained by the cell were with 10 of serum to binding on of Protein in 10 1 were and was for 2 more, at the were further for with the or from both was with of and for in were cultured on for were with in at for and for with in the of both microtubule-associated protein 2 and PIPPin, neurons were with (Roche Molecular and serum or in for 1 at in a The respectively, to and to for in were in an with and by We reported in a previous paper (19Castiglia D. Scaturro M. Nastasi T. Cestelli A. Di Liegro I. Biochem. Biophys. Res. Commun. 1996; 218: 390-394Crossref PubMed Scopus (29) Google Scholar) that PIPPin contains two regions and in with chemical homology to double-stranded RNA-binding more in the that, in the of PIPPin a putative CSD is also The of this domain with present in a number of other as well as nucleic acid-binding proteins that the most conserved portions are the and the region in between these motifs homology to the prokaryotic than to the eukaryotic on the of amino Moreover, according to protein PIPPin would the homology with E. that seems to be a for RNA. in the other eukaryotic however, the CSD of PIPPin is flanked on both sides by putative RNA-binding suggested that PIPPin was an RNA-binding this is the was clearly by the fact that a fusion protein the H3.3 transcribed in from by The an of about that is about more than fusion protein (i.e. about As the has to be to we can that about nucleotides of RNA are The results demonstrated that PIPPin bind RNA. We it any for the encoding H3.3 that, among only H3.3 2) and H1° transcripts, the were to with H3.3 transcripts, present in the binding PIPPin specifically to RNAs encoding H1° and H3.3 histone variants. The of H1° and H3.3 the presence, to the coding regions of the two of a number of with to high in high this of about nucleotides in both the portion of the 3′-UTR, the polyadenylation in the Moreover, the same region is of a that was previously suggested by to be to a highly conserved in H3.3 mRNAs 5Castiglia D. Cestelli A. Scaturro M. Nastasi T. Di Liegro I. Neurochem. Res. 1994; 19: 1531-1537Crossref PubMed Scopus (31) Google Scholar and Fig. 3 To the of this region in binding PIPPin, we and different of the original H1° and H3.3 inserts and used these new plasmids as to synthesize a of As in Fig. 3 all the RNAs that the very end of the of both H1° 3 and H3.3 RNAs all the as H3.3) are to that PIPPin binds specifically to the 3′ end of both H1° and H3.3 as we on the of H1° and H3.3 As a first of PIPPin in we prepared the fusion We used the total serum from or to the expression of PIPPin brain development and its We a the concentration of which at and an level thus a to the reported for PIPPin mRNA (19Castiglia D. Scaturro M. Nastasi T. Cestelli A. Di Liegro I. Biochem. Biophys. Res. Commun. 1996; 218: 390-394Crossref PubMed Scopus (29) Google Scholar). As in the is enriched in the suggesting that PIPPin is to or PIPPin is also present in brain nuclear extracts We if PIPPin is expressed in we prepared neurons from embryonic day 16 rat cerebral cortices, and cultured in a selective, serum-free (22Cestelli A. Savettieri G. Ferraro D. Vitale F. Dev. Brain Res. 1985; 22: 219-227Crossref Scopus (36) Google Scholar) on laminin G. Mazzola G.A. Rodriguez Sanchez M.B. Caruso G. Di Liegro I. Cestelli A. Cell. Mol. Neurobiol. 1998; 18: 369-378Crossref PubMed Scopus (13) Google Scholar). or days of total RNA or nuclear and extracts were prepared from As in PIPPin mRNA is highly expressed in the protein was by in nuclear and extracts from both cultured neurons and and brain and was were used in the the of this that was we used total serum for Fig. is to the results of PIPPin by the protein to both nucleus and in cultured The nuclear is in the present in the of this is in Fig. B and PIPPin the same the clearly and the expected G. 1994; 17: PubMed Scopus Google Scholar). Moreover, the of in the nucleus of the protein in complexes and/or to the nuclear To histone complexes could be in cell we first and to specifically the putative PIPPin-RNA As in Fig. a number of were H3.3 RNA was with 1) or nuclear 2) brain among these however, only a with of about was by B). The of the is about than the PIPPin The in between PIPPin and the putative PIPPin-RNA is thus the same in the of the by the fusion protein for in Fig. the is more in the nuclear 2) than in the PIPPin is present in both in order to this in its RNA binding we that this might be modulated by post-translational the of this we two-dimensional electrophoresis and on both and nuclear cell extracts. results of these As expected, both fractions were clearly after the in the and nuclear PIPPin different PIPPin is present as a with a (i.e. the expected for the other a high proportion or of nuclear PIPPin in a region of the gel that to that a high proportion of PIPPin might be post-translationally modified in the nucleus by in order to bind RNA. the proteins in the brain, variants of both linker as H1°) and core as H3.3) are of as their chromatin further of the potential of the in the absence of DNA and cell is thus most to the of these proteins is regulated in the developing brain and in postmitotic the of mRNA and in control of both in development and (for see Refs. 10Oleynikov Y. Singer R.H. Trends Cell Biol. 1998; 8: 381-383Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, A. Biochem. 1996; PubMed Scopus Google Scholar, and J. Trends Genet. 1996; 12: Full Text PDF PubMed Scopus Google Scholar). The for specific regulation of mRNA in the RNA 1998; 21: Full Text Full Text PDF PubMed Scopus Google Scholar). transcripts of untranslated sequences are binding for a number of RNA-binding proteins. RNA-binding proteins on the at the moment of transcription (16Burd C.G. Dreyfuss G. Science. 1994; 265: 615-662Crossref PubMed Scopus (1734) Google Scholar, 17Siomi H. Dreyfuss G. Curr. Opin. Genet. Dev. 1997; 7: 345-353Crossref PubMed Scopus (232) Google Scholar) and are for the of the (7Matsumoto K. Wassarman K.M. Wolffe A.P. EMBO J. 1998; 17: 2107-2121Crossref PubMed Scopus (177) Google Scholar). the several classes of RNA-binding proteins to proteins called see Refs. P.L. Marahiel M.A. Trends Biochem. Sci. 1998; 23: 286-290Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar and 21Matsumoto K. Wolffe A.P. Trends Cell Biol. 1998; 8: 318-323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) an the of which with both DNA and RNA to control transcription and/or translation of specific genes K. Wolffe A.P. Trends Cell Biol. 1998; 8: 318-323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). the domain, CSD proteins mainly as RNA a that is as it is now clear that RNAs or even in the absence of protein which to be also for the of complexes F. J. D. Nucleic Acids Res. 1998; PubMed Scopus Google Scholar). most however, interesting of PIPPin on the its specificity for the encoding H1° and PIPPin would the homology with E. that seems in fact to be a for its to other eukaryotic is flanked on both sides by other putative RNA-binding and We if of PIPPin on on the two or on the of all these the present is for at two it an of a RNA-binding that is also specific for of it is clear that, in brain, of the histone variants H1° and H3.3 is regulated mainly at the post-transcriptional level M. Cestelli A. Castiglia D. Nastasi T. Di Liegro I. Neurochem. Res. 1995; 20: 969-976Crossref PubMed Scopus (27) Google Scholar). it this control is previous (24Scaturro M. Nastasi T. Raimondi L. Bellafiore M. Cestelli A. Di Liegro I. J. Biol. Chem. 1998; 273: 22788-22791Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) that specific for the H1° are present in the developing We that, in to H1° mRNA can also bind PIPPin, that is to H3.3 mRNA the previous and present results suggest that post-transcriptional regulation of histone variants on a of of which are highly specific for a whereas might by different of the same their this an interesting is that PIPPin binds both H1° and H3.3 RNAs at the very end of the which the putative polyadenylation suggesting that polyadenylation might be in Although we of such a the of PIPPin in both the cytoplasm and the nucleus of nerve that the protein might histone from transcription to as suggested by the of the nuclear in the of histone nuclear Finally, with the of RNA-binding PIPPin in we used the in an to putative H3.3 complexes from nuclear and brain extracts. this might be as it has suggested K.M. Curr. Opin. Struct. Biol. 1997; 7: 336-342Crossref PubMed Scopus (110) Google Scholar) that RNA specific complexes with both as well as that might of the protein by the of other and/or RNA-binding factors might a further these we even if with PIPPin is present in both the cytoplasm and nucleus, the concentration of PIPPin-RNA complexes is in the nuclear extracts. order to this we are to that the protein in the nucleus. As are protein and potential in its amino we that nuclear RNA binding activity might on PIPPin phosphorylation. was by the that a proportion of nuclear PIPPin is more acidic than it is that PIPPin must be in order to bind RNA with high Although the that PIPPin activity might be modulated by post-translational is it that histone binding the of this binding on histone mRNA might be in by such as and in the rate of histone variant and of their into chromatin might of the organization and the transcription potential of We M. L. and M. C. for and D. for with