The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

Abstract

Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.

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+…”

“…The xSLBP1 and xSLBP2 proteins were divided into three domains, based on the definition of the 73 amino acids in the center of the protein as an RNA-binding domain (Wang et al+, 1996c)+ We use the term RBD for the RNA-binding domain; this term simply reflects the ability of the 73-amino acid domain to bind RNA and is not meant to imply any similarity in structure to other RNA-binding proteins+ This 73-amino acid region binds the stem-loop with an affinity similar to full-length SLBP (Wang et al+, 1996c)+ The amino terminal and the carboxyl terminal regions were arbitrarily designated as other domains of the SLBPs+ The amino acid sequences of the RBD of xSLBP1 and xSLBP2 are compared in Figure 2A+ The terminal amino acids at each end of the RBD are conserved in both Xenopus SLBPs and in the mammalian SLBP+ Only minimal changes were introduced at the amino terminal boundary of the domain during construction of the various chimeric proteins (Fig+ 2B,C)+ Each construct was transcribed with SP6 RNA polymerase to produce a synthetic capped polyadenylated mRNA that was injected into stage VI oocytes+ Expression of the protein product was detected by its ability to bind a radiolabeled histone mRNA stem-loop (Williams & Marzluff, 1995)+ The oocytes were subsequently injected with the mouse histone H2a-614 gene and the ability of the protein(s) to affect histone pre-mRNA processing determined+…”

“…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

“…A comparison of the consensus sequence of the histone mRNA 39 stem-loop with the specificity determinants for SLBP recognition+ Shown in gray boxes are nucleotides that show a deleterious effect on SLBP affinity when mutated or deleted+ and orient it for cleavage by the U7 snRNP+ We found, however, that much of the 39 flanking region could be deleted without a large effect on the affinity of the SLBP-RNA interaction (Fig+ 4)+ Deletion of four of the residues had only a slight effect, and deletion of the entire 39 flanking region increased the K d 5+5-fold+ This suggests that SLBP is not making extensive contacts to most of this region, but rather is only interacting with the first A residue 39 of the stem+ Alternatively, the presence of a 39 overhanging adenosine has been shown to contribute approximately 1 kcal/mol to the stability of short oligonucleotide duplexes (Freier et al+, 1986)+ It is possible, therefore, that A22 simply serves to stabilize the stem and is not contacting SLBP+ These data suggest that SLBP may enhance 39-end processing by direct or indirect recruitment of the U7 snRNP to the cleavage site as has been previously proposed (Dominski et al+, 1999), rather than by directly ordering the RNA structure at the cleavage site+ The sequence conservation of the 39 flanking region (and possibly the bottom G-C pair of the stem) may be necessary for the binding of other components of the 39-end processing machinery+ SLBP has no homology with any other proteins thus far discovered+ It contains a relatively small, approximately 73 amino acid, RNA-binding domain predicted to contain three a-helices (Wang et al+, 1996;Martin et al+, 2000)+ Previously characterized RNA-binding domains (Draper, 1999) recognize either the loop in a sequence-specific manner, as with U1A (Oubridge et al+, 1994), or the loop and end of a stem by relying on noncanonical base pairs or bulges to insert helices into the major groove, as in the bacteriophage lambda N peptide (Legault et al+, 1998)+ Others recognize WatsonCrick A-form RNA helices in a predominantly sequenceindependent manner (Ryter & Schultz, 1998) …”

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+…”