H10 RNA-binding Proteins Specifically Expressed in the Rat Brain

Abstract

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 posttranscriptional regulatory components. In particular, the expression of H1 0 in rat cortical neurons is regulated mainly at the post-transcriptional level, and unknown cellular proteins are likely to affect H1 0 mRNA stability and/or translation. In looking for such factors, we tested the ability of rat brain extracts to protect H1 0 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 H1 0 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 H1 0 mRNAs. These findings suggest that the proteins identified play a critical role in regulating the expression of H1 0 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 nuclea...

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

“…The observed differences are not due to modifications of gene transcription (24) and should depend on post-transcriptional regulation. The search of RBPs able to bind H1˚-and/or H3.3-mRNA, and possibly involved in their metabolism, in the past led to the identification of three H1˚ RNA-binding proteins (p40, p70 and p110) (11) and to cloning of an H3.3/H1˚ RNA-binding protein (PIPPin, also known as CSD-C2), which contain a cold-shock domain (13). More recently, a second protein has been cloned through an experimental approach based on screening of an expression cDNA library by a functional binding assay with a labeled, in vitro transcribed histone RNA (15).…”

“…About 0.5x10 6 cpm (0.5-2.0x10 7 cpm/pmol of RNA) of purified H1˚ RNA were mixed with recombinant protein (50 ng), prepared as described above. For the T1 protection assay, we used a previously described method (11) except that cross-linking of RNA to proteins was performed before incubation with T1 RNase (EC 3.1.27.3; Roche). RNA-protein complexes were analyzed by denaturing electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide slab gel (PAGE).…”

“…Among post-transcriptionally regulated genes, those encoding histone variants, such as H1˚ (6)(7)(8) and H3.3 (2,9,10) are of interest for the possible involvement of histone variants in the replication-independent chromatin remodelling induced by extracellular stimuli (2). In looking for proteins able to bind mRNAs encoding the histone variants H1˚ and H3.3, we identified a set of proteins that bind the one and/or the other histone mRNAs (11). We also cloned a cDNA that encodes a protein, that we called PIPPin (12)(13)(14), now also known as CSD-C2, that contains a cold shock domain and binds both mRNAs at the level of the polyadenylation signal (13).…”

RNA‐binding ability of PIPP in requires the entire protein

“…19) was linearized by restriction and used as template to synthesize 32 P-labelled H1° mRNA, from the T3 RNA polymerase promoter, according to Promega instructions. T1 protection assays were carried out as described elsewhere [20]. Protein-RNA complexes were analysed by electrophoresis on denaturing 15% polyacrylamide and autoradiography, as described [20].…”

“…T1 protection assays were carried out as described elsewhere [20]. Protein-RNA complexes were analysed by electrophoresis on denaturing 15% polyacrylamide and autoradiography, as described [20].…”