The gene for histone RNA hairpin binding protein is located on human chromosome 4and encodes a novel type of RNA binding protein

Abstract

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic 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.

Dual role for the RNA-binding domain of Xenopus laevis SLBP1 in histone pre-mRNA processing

“…The 39 end of histone mRNA and its bound SLBP play a critical role in multiple aspects of histone mRNA metabolism, including pre-mRNA processing (Dominski et al+, 1995;Wang et al+, 1996c;Martin et al+, 1997), export (Eckner et al+, 1991;Williams et al+, 1994), translation (Sun et al+, 1992;Gallie et al+, 1996), and mRNA stability (Pandey & Marzluff, 1987;Williams et al+, 1994)+ The frog oocyte is a convenient experimental system to study the many aspects of RNA metabolism because one can express or inject both specific RNAs and proteins and follow their subsequent metabolism+…”

“…Replication-dependent histone mRNAs are not polyadenylated; they end in a highly conserved 26-nt sequence containing a 16-nt stem-loop (Marzluff, 1992;)+ Histone pre-mRNAs do not contain introns, and only a single endonucleolytic cleavage to form the 39 end (Gick et al+, 1986) is necessary to generate a mature histone mRNA+ This cleavage requires two cis-acting sequences in the histone pre-mRNA: the stem-loop and a purine-rich histone downstream element (HDE), which is located approximately 10 nt past the cleavage site+ These elements are recognized by two trans-acting factors: the stemloop binding protein (SLBP) (Wang et al+, 1996c;Martin et al+, 1997) and the U7 snRNP+ The 59 end of U7 snRNA base pairs with the HDE (Mowry & Steitz, 1987;Cotten et al+, 1988;Soldati & Schümperli, 1988)+ Additional factor(s) that have not been well characterized, including a heat-labile factor (Gick et al+, 1987), are also required+ One role of the SLBP in the processing reaction is stabilization of binding of the U7 snRNP to the histone pre-mRNA (Spycher et al+, 1994;, resulting in a stable complex containing histone pre-mRNA, SLBP, and U7 snRNP+…”

The stem-loop binding protein forms a highly stable and specific complex with the 3′ stem-loop of histone mRNAs

“…Replication-dependent histone mRNAs contain a 26-nt stem-loop structure at their 39 terminus+ The sequence of the stem-loop, as well as the sequence immediately flanking the stem-loop, is highly conserved among all metazoans+ SLBP is found associated with this stemloop in both the nucleus and cytoplasm of higher eukaryotes (Mowry et al+, 1989;Vasserot et al+, 1989;Pandey et al+, 1991;Hanson et al+, 1996;Martin et al+, 1997)+ Mutations that disrupt formation of the SLBP-RNA complex in nuclear extract interfere with proper pre-mRNA processing, as well as mature mRNA localization and cell-cycle dependent stability (Pandey & Marzluff, 1987;Harris et al+, 1991;Sun et al+, 1992;Streit et al+, 1993;Williams et al+, 1994;Dominski et al+, 1995)+ Here we have used direct K d measurements with purified protein and RNA components to define the affinity and specificity of the SLBP-histone mRNA interaction+ Nitrocellulose filter-binding experiments were performed to determine the affinity of SLBP for the RNA stem-loop+ This interaction was found to be quite tight, with a K d ϭ 1+5 nM, corresponding to a ⌬G of Ϫ12+0 kcal/mol+ The measured affinity was strongly affected by ionic strength, dropping off quickly with increasing concentrations of MgCl 2 or KCl (Fig+ 2)+ This suggests that electrostatics play a key role in recognition of the stem-loop by SLBP, similar to what is commonly observed for many complexes involving protein recognition of a simple DNA duplex (Misra et al+, 1994)+ However, even at very high salt concentration (800 mM KCl), binding was still relatively tight (60 nM), suggesting that hydrophobics contribute to the interaction, perhaps through recognition of the loop or flanking nucleotides+ As observed previously in extracts, mutations in the 6-bp stem greatly reduced SLBP binding (Fig+ 3A)+ The bottom 2 bp are invariantly G-C base pairs+ Previous work had shown a large drop in binding affinity upon transversion of the bottom two G-C base pairs together (Williams & Marzluff, 1995)+ We found that transversion of the bottom base pair alone had no detectable effect on binding, whereas transversion of the second base pair (G7-C20) led to a marked increase in the K d + In addition, we found that mutation of the third base pair, always a pyrimidine-purine pair, to G-C also had a strong, negative effect on binding+ Similarly, mutation of the invariant U-A pair at the top of the stem had a dramatic deleterious effect on SLBP binding+ Taken together, these data suggest multiple SLBP contacts to the RNA stem, particularly to base pairs G7-C20, C8-G19, and U11-A16 (Fig+ 5)+ Some of these effects could result from subtle changes in the helical parameters that reposition specificity determinants in the loop relative to the base of the stem or flanking regions+…”

“…Most eukaryotic mRNAs are polyadenylated at their 39 ends, providing a binding site for several nuclear and cytoplasmic protein factors+ These factors control the transport of mRNA from the nucleus, targeting to polyribosomes for translation, and regulation of message stability+ The replication-dependent histone mRNAs are the only RNA polymerase II transcripts that lack this poly(A) tail+ Instead, the histone pre-mRNAs contain two highly conserved regions at their 39 terminus: a 26-nt stem-loop structure followed by a purine-rich sequence known as the histone downstream element (HDE; Birnstiel et al+, 1985;Marzluff, 1992)+ These premRNAs are processed in the nucleus by a single endonucleolytic cleavage approximately 5 nt downstream of the stem-loop, catalyzed by the U7 snRNP through base pairing of the U7 snRNA with the HDE (Gick et al+, 1986)+ After processing, the mature messages are exported from the nucleus to the cytoplasm, where they are targeted to polyribosomes and translated (Fig+ 1; Eckner et al+, 1991;Sun et al+, 1992;Williams et al+, 1994)+ These events are tightly coupled to the cell cycle, resulting in high histone mRNA levels immediately preceding DNA replication+ This posttranscriptional regulation is responsible for the majority of the cell-cycle dependent control of histone mRNA levels (Schumperli, 1986;Marzluff & Pandey, 1988;Harris et al+, 1991)+ The stem-loop binding protein (SLBP), a 32-kDa protein with no known homologs, is associated with the stem-loop at the 39 end of the histone messages in both the nucleus and cytoplasm of higher eukaryotes (Fig+ 1; Mowry et al+, 1989;Vasserot et al+, 1989;Pandey et al+, 1991;Hanson et al+, 1996;Martin et al+, 1997)+ SLBP levels are cell-cycle regulated, being highest during S-phase when histone mRNA levels are increased (Whitfield et al+, 2000)+ SLBP is necessary for efficient 39 end processing of histone pre-mRNA by the U7 snRNP (Streit et al+, 1993;Dominski et al+, 1995)+ In addition, mutations in the stem-loop that disrupt formation of the SLBP-RNA complex result in the retention of the mRNA in the nucleus and failure to target to polyribosomes (Sun et al+, 1992;Williams et al+, 1994)+ These same mutations also disrupt the cell-cycle dependent regulation of histone mRNA stability (Pandey & Marzluff, 1987; Harris et al+, 1991)+ Through these mecha-nisms, it is believed that formation and maintenance of the SLBP-RNA complex contributes to the cell-cycle regulation of histone levels in higher eukaryotes+ Formation of the SLBP-histone mRNA complex has been previously studied using competition assays in nuclear extract …”

Identification of RNAs that bind to a specific protein using the yeast three-hybrid system

“…The yeast three-hybrid system (SenGupta et al+, 1996) detects the interaction between an RNA and a protein+ In this system, a transcription factor is assembled by the use of an RNA bridge that brings a fusion protein containing a DNA-binding domain together with a fusion protein containing an activation domain+ The assembly of this ternary complex depends on each fusion protein containing an RNA-binding domain that binds to a site in the RNA molecule+ As fixed components, the DNA-binding domain hybrid is the LexA protein fused to the bacteriophage MS2 coat protein, and the hybrid RNA contains two copies of the coat protein binding site+ The three-hybrid system has been used to identify proteins that bind to RNA sequences such as the 39 end of histone mRNA (Wang et al+, 1996;Martin et al+, 1997) and an element in the 39 untranslated region of the Caenorhabditis elegans fem3 mRNA (Zhang et al+, 1997), and to detect and analyze known RNA-protein interactions (e+g+, Bacharach & Goff, 1998) + We have now adapted this system to identify RNA ligands of an RNA-binding protein+ We generated an RNA expression library of genomic sequences from the yeast Saccharomyces cerevisiae fused to coat protein binding sites, and screened it for RNAs that can bind the yeast Snp1 protein, a homolog of the human U1-70K protein+ This search identified an RNA fragment containing the loop I sequence of SNR19 RNA (U1 RNA), whose binding to Snp1 has been previously demonstrated by in vitro assays (Kao & Siliciano, 1992)+ Additionally, the search identified other Snp1-binding RNAs that yielded a weak signal in this assay, as well as RNA sequences that alone can activate transcription when bound to the promoter of a reporter gene+…”