H10 RNA-binding Proteins Specifically Expressed in the Rat Brain*

During brain maturation, histone H10 accumulates in both nerve and glial cells. The expression of this “linker” histone, the role of which still remains unclear, is a complex process, having both transcriptional and post-transcriptional regulatory components. In particular, the expression of H10 in rat cortical neurons is regulated mainly at the post-transcriptional level, and unknown cellular proteins are likely to affect H10 mRNA stability and/or translation. In looking for such factors, we tested the ability of rat brain extracts to protect H10 RNA probe from degradation by T1 RNase. The results reported here demonstrate that rat brain contains at least one major (p40) and two minor (p110 and p70) binding factors, specific for H10 RNA, all of which are much more or exclusively expressed in adult rat brain, when compared with other tissues. The binding of the factors is confined to a portion of the 3′-untranslated region (3′-UTR), which is highly conserved among murine and human H10 mRNAs. These findings suggest that the proteins identified play a critical role in regulating the expression of H10 histone in the brain of mammals.

During development and differentiation, cells undergo precise sequences of events that imply switching on or off different sets of genes, and even terminally differentiated cells respond to ever changing environmental conditions by modulating the transcriptional activity of many genes. As this often implies the need for remodeling chromatin (for review, see Ref. 1), it is not surprising that non-allelic isotypes of histones are synthesized in differentiated cells and enter chromatin, probably at topologically defined regions of the nucleus.
The H1 class of histones, also known as linker histones, are involved in organizing chromatin higher structures as well as in regulating specific gene expression (for discussion, see Ref. 2). In addition to a number of other pre-existing H1 subtypes, histone H1 0 appears in cells, during terminal differentiation (3,4) or after growth inhibition (5,6). Although the role of this linker histone remains unclear, it has been proposed to localize specifically to the periphery of nucleus (7). Like other replacement histones, H1 0 accumulates in postmitotic cells and is synthesized in the absence of DNA replication (8 -11); however, transcription of the H1 0 gene seems to be a replication-dependent event, at least in some cell types (12,13). Regulation of H1 0 expression is thus likely to be a complex process with both transcriptional and post-transcriptional components.
In maturing brain, H1 0 accumulation was demonstrated in both neurons (11, 14 -16) and glial cells (9). However, the role of H1 0 in gene expression remains unclear. We previously cloned two cDNAs encoding rat histones H1 0 (17) and H3.3 (14), respectively. We used these cDNAs as probes to study the accumulation of the corresponding messages during rat brain development (14) and in cultured neurons (18). The effects of transcriptional inhibition by actinomycin D and the results of nuclear run-on experiments suggested that expression of both H1 0 and H3.3 is regulated mainly at the post-transcriptional level (18). Post-transcriptional control processes often include regulation of mRNA localization, stability, and translation (18 -31) and are mediated by several RNAbinding proteins (22,26,(32)(33)(34)(35)(36)(37)(38)(39)(40). Therefore, it is likely that cellular factors, possibly expressed differentially in development, are involved also in histone mRNA binding and regulation in the brain.
The present study aimed, in particular, at the identification of proteins able to bind the mRNA encoding H1 0 histone. We identified three brain-specific proteins that bind H1 0 RNA probe with high specificity.

EXPERIMENTAL PROCEDURES
Preparation of Tissue Extracts-Fresh tissues from developing or adult rats were homogenized in nuclei buffer (NB: 0.32 M sucrose; 50 mM sodium phosphate buffer, pH 6.5; 50 mM KCl, 0.15 mM spermine; 0.15 mM spermidine; 2 mM EDTA and 0.15 mM EGTA), containing protease inhibitors (2 g/ml aprotinin, 2 g/ml antipain, 2 g/ml leupeptin, 2 g/ml pepstatin A, 1.0 mM benzamidine, and 1.0 mM phenylmethylsulfonyl fluoride, Sigma) and centrifuged at 1,000 ϫ g for 10 min at 4°C. The supernatant was then used as such (post-nuclear extracts) or further centrifuged at 10,000 ϫ g for 10 min to collect mitochondria. In the latter case, the new supernatant was finally centrifuged at 100,000 ϫ g for 60 min to separate the microsomal from the postmicrosomal (S-100) cell fraction. All the fractions, in NB, were split into aliquots and rapidly frozen in liquid nitrogen. Protein concentration was determined according to Lowry et al. (41).
Preparation of in Vitro Transcripts-The plasmids pMH1 0 (17), pDH3 (obtained by ligation of the 5Ј region of pDH 33-2-and the 3Ј region of pDH 33-1-inserts, described in Ref. 14; it contains the entire sequence encoding the 1.2-kilobase H3.3 mRNA from rat) and pA1.3K (42) were linearized by restriction with BamHI (pMH1 0 ) or HindIII (pDH3 and pA1.3K) and used as templates for in vitro transcription of both cold and 32 P-radiolabeled H1 0 , H3.3, and c-erbA␣2 transcripts, respectively, from the T3 (pMH1 0 ) or the T7 (pDH3 and pA1.3K) RNA polymerase promoters (all the buffers and enzymes used for transcription were purchased from Promega). RNA was extracted once with phenol and twice with chloroform and precipitated with ethanol and sodium acetate (0.3 M final concentration). The transcripts were collected by centrifugation at 10,000 ϫ g for 15 min, washed in 75% ethanol, and resuspended in distilled water. Small aliquots were used for counting and/or analysis on denaturing gels. On the basis of these analyses, we calculated the transcript concentration to be used in the next analyses.
T1 Nuclease Protection Assay-T1 nuclease protection assay was carried out, with modifications, according to the method described by Zaidi and Malter (43) and modified by Izquierdo and Cuezva (44). Briefly, cell extracts (10 -15 g) were incubated for 10 min, at room temperature, with 0.5-1.0 ϫ 10 6 cpm (specific activity: 0.5-2.0 ϫ 10 7 cpm/pmol of RNA) of radiolabeled RNA, transcribed in vitro. Samples * This work was supported by the Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
were then incubated for 30 min at 37°C, with 100 units of T1 RNase (EC 3.1.27.3) to degrade the whole of the RNA except the portions protected by bound proteins; the extracts were finally exposed to a Spectroline UV (254 nm) lamp (Aldrich Chemical Co., Inc.) for 15-20 min, in ice bath, to cross-link RNA to proteins. The covalent radioactive complexes were analyzed by denaturing electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide slab gels (PAGE), 1 according to Laemmli (45), and the gels were directly exposed to x-ray film for autoradiography, with intensifying screens, for 12-18 h, at Ϫ70°C.
At the end of exposure, the gels were stained with Coomassie Brilliant Blue R-250 (Sigma) to confirm the loading of equal amounts of proteins per lane.
Affinity of Binding-To quantify the affinity of the factors for RNA, identical amounts (15 g) of post-nuclear lysates from brain at the 18th day of embryonal development (E18) were incubated with increasing amounts of radioactive 1300-nt-long transcript. Samples were then treated and analyzed as described above. After electrophoresis, the gel was exposed in the same cassette with a piece of nylon membrane on which known amounts of the same RNA, used for the binding reaction, had been spotted. After variable times of exposure, the film was scanned in a HP Scan jet 4C/T and analyzed by the Sigmagel program, version 1.0 (Jandel Scientific). The planimetries of scans were finally used to calculate concentrations of RNA. To compare the intensities of bands corresponding to protein-RNA complexes with the known amounts of RNA, we substituted concentration of radioactive incorporated uridine (U) for RNA concentration (each molecule of the 1300-ntlong H1 0 RNA fragment contains 307 U residues) and assumed an arbitrary size of 40 nt (with an average of 10 U residues) for the fragment of RNA covalently bound to p40. Briefly, from the intensity of each band, it was calculated how many femtomoles of a 10 U-containing RNA fragment were bound; this number was assumed as the number of protein-RNA complexes per 15 g of protein. Fig. 1 shows representative results of T1 RNase protection assays in which H1 0 and H3.3 in vitro transcripts were incubated with equal amounts (10 g) of post-nuclear extracts from E18 rat brain. H1 0 RNA binds to one major (40 kDa: p40) and two minor (about 110 and 70 kDa, respectively: p110 and p70) proteins; these bands are clearly different, both for position and intensity, from those visible in the case of H3.3 RNA. Specificity of the factors identified was confirmed by experiments in which unlabeled H1 0 RNA (25-fold excess) but not other brain-specific RNAs (such as H3.3 and c-erbA␣2) were able to abolish the radioactive bands (Fig. 2).

Identification of H1 0 RNA-binding Factors-
The H1 0 RNA-binding proteins were present throughout the investigated period of brain development (Fig. 3A), and they were richest in the brain, compared with other tissues, in adult animals (Fig. 3B). In the adult brain, a fourth protein (of about 90 kDa) is sometimes visible (see the arrowhead in Fig. 3B).
The presence of this protein might relate either to aging or to individual variation; however, we have not yet found any clear correlation between this protein and the physiological state of the animal.
Concerning localization of the factors identified, we found that the major band was enriched in the microsomal fraction, whereas the minor ones were more concentrated in the cytosolic S-100 fraction (Fig. 4).
Identification of RNA Regions Involved in Binding to the Factors-The first step toward the identification of the RNA region involved in binding was to test the binding capacity of two RNAs corresponding, respectively, to the first 1300 and to the last 411 nucleotides of the H1 0 RNA (Fig. 5). As shown in Fig. 6A, only the larger RNA (1-1300, in the figure) was able to bind to all the factors. This finding was also confirmed by experiments in which the binding of the whole H1 0 RNA was competed by a 10-fold excess of the longer RNA but not by the shorter one (1301-1711) (Fig. 6B).
Then we tested the binding capacity of three smaller RNAs corresponding to three different regions of the 1300-nt-long RNA: a) the coding region (nt 1-496); b) from nt 497 to nt 900 1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; nt, nucleotide(s); UTR, untranslated region.
FIG. 1. T1 RNase protection assay of radiolabeled H1 0 or H3.3 RNA, transcribed in vitro and incubated with post-nuclear extracts from E18 developing brain. Brain extracts (10 g) were incubated with 1.0 ϫ 10 6 cpm of radiolabeled H1 0 or H3.3 RNA probe, for 10 min at room temperature; samples were then incubated for 30 min at 37°C with 100 units of T1 RNase and exposed for 20 min, in ice bath, to UV radiation (254 nm) as described under "Experimental Procedures." RNA-protein complexes were analyzed by 15% SDS-PAGE, and the gel was directly exposed to x-ray film for 16 h, at Ϫ70°C.  3 and 4) recognized the proteins. Scanning of the autoradiographies revealed that the intensity of the 40-kDa band bound to the entire 1300-nt-long RNA (lane 1) is equal to the sum of the intensities of the corresponding 40-kDa band detected with the two smaller RNAs (lanes 3 and 4). This finding suggests that p40 has binding sites for both regions of 3Ј-UTR of the H1 0 mRNA.
Binding Affinity-On the basis of the intensity of the spots reported in Fig. 8A (known amounts of the 1300-nt-long RNA: see "Experimental Procedures") we calculated the amount of RNA bound to p40 (Fig. 8B). As the RNA concentration in the binding mixture was also known, we could calculate the apparent K D of the complexes by Scatchard analysis (Fig. 8C). K D values thus calculated, from different experiments, are in the range of 20 -30 nM. As described under "Experimental Procedures," we assumed an arbitrary length of 40 nt for the bound RNA. This size roughly represents an upper limit for the real, and still unknown, length of the sequence protected by p40: in the absence of cross-linking treatment, we did not notice, indeed, any radioactive band after denaturing electrophoresis on gels as concentrated as 15% polyacrylamide. On the contrary, Partially purified cell subfractions (10 g each), from E18 fetal brain, were incubated with 0.5 ϫ 10 6 cpm of radiolabeled H1 0 RNA. Pn, postnuclear; Mit, mitochondrial; Micr., microsomal; S100, postmicrosomal; Ϫ, without cell fraction. p40-bound RNA might be shorter than 40 nt and/or contain less than 10 U residues. Under these latter hypotheses, the number of protein-RNA complexes formed at a given RNA concentration could be underestimated, and the real K D values would be smaller than those reported here. DISCUSSION The generation of specific neuronal phenotypes depends on the synthesis and intracellular localization of specific regulatory as well as structural proteins. On the basis of several studies, it appears that the fine control of these aspects of protein metabolism depends largely on post-transcriptional regulation of the metabolism of the corresponding mRNAs (29, 46 -53). Among the gene products whose concentration changes during neural cell maturation, differentiation-associated variants of both linker (such as H1 0 ) and core histones (such as H3.3) are of great interest, as their entering chromatin may further affect the transcriptional potential of the genome (54,55).
In maturing brain, H1 0 accumulation seems to be regulated mainly at the post-transcriptional level (18). As increasing evidence points to the importance of RNA-binding proteins in the post-transcriptional control of gene expression in brain (46 -51), we used a slightly modified version of the T1 RNase protection assay described by other authors (43,44), with the goal of identifying RNA-binding proteins that are expressed in developing brain and specific for the mRNA encoding H1 0 . This approach allowed us to identify one major (p40) and two minor (p70 and p110) factors that bind to H1 0 mRNA with high specificity. Because the expression (and/or the binding ability) of these factors does not change significantly from E18 to adulthood, they are probably requested continuously both in differentiating and mature brain. In particular, one or more of these factors might be directly involved in enhancing H1 0 mRNA translation and degradation. This latter function would be consistent with the need of maintaining low concentrations of H1 0 mRNA, without inhibiting transcription of the H1 0 gene. Interestingly, the factors described here are predominantly or exclusively expressed in the brain. This suggests that the function of these proteins is linked to a pathway of H1 0 gene regulation specific to the brain. Furthermore this brain-specific function might have been conserved during evolution of mammals, as the factors identified recognize, in the 3Ј-UTR of H1 0 RNA, regions that contain blocks of sequences highly conserved among murine and human H1 0 genes (17).
It should be also noted that one (p40) of the H1 0 mRNAbinding factors is enriched in the microsomal subcellular fraction, while the other two proteins remain in the cytoplasm. Presumably, recruitment of H1 0 mRNA to ribosomes, its translational activation, and perhaps, enhanced degradation are functions managed by a complex formed by both soluble and anchored proteins. Anchoring the complex to membranes (possibly to the nuclear envelope) and/or to the cytoskeleton might have obvious advantages for localizing H1 0 mRNA to cytoplasmic regions from which newly synthesized H1 0 histone might easily reach specific chromatin domains.
Finally, it should be stressed that, to our knowledge, the factors identified in the present work represent the first example of brain-specific RNA-binding proteins that bind to a highly conserved region of one specific mRNA. The fact that this mRNA is the one encoding H1 0 protein adds interest to the finding, as further understanding of regulation of H1 0 synthesis might help to shed light on the role that this linker histone plays in chromatin remodeling in postmitotic cells.