Stem-loop binding protein facilitates 3'-end formation by stabilizing U7 snRNP binding to histone pre-mRNA

Abstract

The 3' end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3'-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3' processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP.

FIG. 1

Effect of altering the affinity of the pre-mRNA for SLBP. (A) Each of the six mutant stem-loops shown was introduced into the mouse histone H2a-614 pre-mRNA. The radiolabeled synthetic pre-mRNAs were incubated for 30 min in a nuclear extract prepared from mouse myeloma cells, as described in Materials and Methods. The RNA was purified, resolved by gel electrophoresis, and detected by autoradiography. The input pre-mRNA (top band) and the shorter cleavage product (bottom band) are indicated. (B) Sequence of 26 nucleotides encompassing the stem-loop structure in the various mutants. (C) Thirty-nucleotide RNAs were synthesized by T7 RNA polymerase with the appropriate oligonucleotide templates. Each RNA consists of 26 nucleotides encompassing the stem-loop structure shown and the GCCC sequence at the 5′ end facilitating synthesis of RNA by T7 polymerase. The 30-nucleotide RNA containing the wild-type stem-loop structure was labeled at the 5′ end with [γ-³²P]ATP and used to detect SLBP in the nuclear extract (NE) by using a mobility shift assay. The samples were analyzed on a 7% polyacrylamide gel under nondenaturing conditions. Unlabeled 30-nucleotide RNA competitors containing the wild-type or mutant stem-loop sequences were added to the reaction at a molar excess, as indicated above each lane. Lanes 2 and 9, no competitor added; lane 1, probe.

FIG. 2

Comparison of H2a-614 and H1t pre-mRNAs as processing substrates. (A) The sequences of the H2a and H1t pre-mRNAs encompassing the stem-loop and HDE are shown. The sequence of the 5′ end of the U7 snRNA and its potential to form base pairs with the HDE are depicted below each pre-mRNA substrate. Watson-Crick base pairs are indicated by vertical lines, and GU base pairs are indicated by dots. (B) The 86-nucleotide H2a-614 pre-mRNA substrate was incubated in a nuclear extract (NE) for 60 min under processing conditions (lane 1). RNA oligonucleotides containing the sequence of either the mutant HDE (HDE⁻) (see Fig. 5A), the H2a-614 HDE, or the H1t HDE were added to the reaction samples at a 100-fold molar excess relative to the substrate (lanes 2, 3, and 4, respectively). The RNA was purified, resolved by gel electrophoresis, and detected by autoradiography. (C) The in vitro processing reaction was carried out with either the H2a-614 (top panel) or H1t (bottom panel) pre-mRNAs under standard conditions (lane 1) or in the presence of a 100-fold molar excess of the competing 30-nucleotide RNAs shown in Fig. 1B, as indicated above each lane (lanes 2 to 5).

FIG. 3

Depletion of SLBP from the nuclear extract and complementation with recombinant SLBP. (A) The 30-nucleotide radiolabeled wild-type stem-loop RNA (lane 1) was incubated in a nuclear extract (NE), and the complex was resolved by gel electrophoresis (lane 2). Lane 3, nuclear extract plus a 100-fold excess of unlabeled competitor 30-nucleotide stem-loop RNA; lane 4, extract treated with preimmune serum (mock depletion); lane 5, extract depleted with anti-SLBP antibody; lane 6, anti-SLBP-depleted extract supplemented with 50 ng of recombinant human SLBP. (B) The same extracts as those in panel A were used in processing reactions with the histone H2a-614 pre-mRNA (top panel) or the H1t pre-mRNA substrates (bottom panel). The RNAs were purified, resolved by electrophoresis in a denaturing polyacrylamide gel (8%; 7 M urea), and detected by autoradiography. Processing in the nuclear extract (NE) under standard conditions and in the presence of a 100-fold excess of 30-nucleotide competitor RNA with wild-type stem-loop sequence is shown in lanes 1 and 2, respectively. Lane 3, mock-depleted extract (preimmune serum); lane 4, extract depleted with anti-SLBP antibody; lane 5, anti-SLBP-depleted extract plus recombinant human SLBP. (C) Processing of the histone H2a-614 pre-mRNA (top panel) or the H1t pre-mRNA (bottom panel) in the nuclear extract depleted of SLBP with biotinylated RNA oligonucleotide containing the wild-type stem-loop. Lane 1, mock-depleted extract (nonspecific biotinylated oligonucleotide); lane 2, extract depleted with biotinylated stem-loop RNA; lane 3, depleted extract complemented with 50 ng of recombinant human SLBP. (D) Equal amounts (50 μg of protein) of the nuclear extracts (NE) used in panels B and C were resolved by electrophoresis on a 12% polyacrylamide–SDS gel, and SLBP was detected by Western blotting. The top band represents the intact 45-kDa SLBP, and the bottom band is a proteolytic cleavage product lacking part of the N terminus of the protein.

FIG. 4

Regions of human SLBP required for histone pre-mRNA processing. (A) The restriction map of the human SLBP cDNA with the boundaries of the three regions of the protein corresponding to the N-terminal domain (N-ter), the RNA binding domain (RBD), and the C-terminal domain (C-ter) is outlined at the top. The constructs are named according to the number of amino acids deleted from the N or C terminus. The hSLBP/20aa has the 20 amino acids immediately after the RNA binding domain (196 to 215) changed to the 20 amino acids present at this position in frog XSLBP2, an SLBP which does not function in pre-mRNA processing (43). The ability of the mutant SLBP to restore processing in the SLBP-depleted extract is summarized to the right of each construct. (B) The nuclear extract (lane 1) was depleted of SLBP with anti-SLBP antibody and was assayed for the ability to cleave the H1t pre-mRNA when complemented with 50 ng of the indicated mutant human SLBP. Depletion of the extract resulted in an almost complete loss of processing activity (lane 2) which was fully restored when the depleted extract was supplemented with the full-length human SLBP (lane 3). Complementation of the depleted extract with the mutant proteins is shown in lanes 4 to 8. (C) Processing activity of the nuclear extract (NE; differing from that in Fig. 1B) before and after immunodepletion is shown in lanes 1 and 2, respectively. Fifty nanograms of human SLBP (lane 3) or mutant proteins was added to the depleted extract, as indicated (lanes 4 to 6). (D) The recombinant proteins were tested for their ability to bind to the stem-loop RNA oligonucleotide. Fifty nanograms of the recombinant protein indicated at the top of each lane was incubated with the radiolabeled stem-loop RNA, and the complexes were resolved by native gel electrophoresis.

FIG. 5

Formation of a stable complex containing the SLBP and the U7 snRNP on the pre-mRNA substrate. (A) Sequences of the substrates used for assaying U7 snRNP binding. The sequence of the parental H2a pre-mRNA comprising the stem-loop structure, the cleavage site represented by an arrow, and the U7 binding site are shown at the top. The nucleotides at the 5′ and 3′ ends of the RNA encoded in the pGEM3 vector are not included. The nucleotide substitutions introduced into the H2a pre-mRNA in order to generate the other pre-mRNA substrates are shown below the H2a sequence. The unchanged sequences are represented by solid lines. The RS mutant of the H2a-614 pre-mRNA contains reversed sequence of the stem-loop structure, as shown in Fig. 1B. (B) The H2a/4G (lane 1) and HDE⁻ (lane 2) substrates shown in panel A were incubated in a nuclear extract under standard processing conditions. The RNA was analyzed as described in the legend to Fig. 2. (C) The H2a-614 substrate (lanes 2 to 6) and the indicated mutant substrates (lanes 7 to 10) were briefly incubated in nuclear extract to allow the formation of processing complexes. The complexes were immunoprecipitated with anti-SLBP antibody, RNA prepared, resolved by electrophoresis on 8.5% polyacrylamide gel containing 7 M urea, and assayed for the presence of U7 snRNA by Northern blotting. Immunoprecipitation was carried out in the presence of the H2a-614 pre-mRNA (lanes 2 to 6) or mutant pre-mRNAs, as indicated (lanes 7 to 10). Lane 1, no substrate added; lane 3, 10 μg of antigenic peptide was added to the reaction mixture prior to addition of the antibody; lanes 4 and 5, immunoprecipitation in the presence of 0.5 μg of a nonspecific 2′-O-methyl oligoribonucleotide or 2′-O-methyl oligoribonucleotide complementary to the 5′ end of U7 snRNA, respectively. Lane 6, immunoprecipitation in the presence of a 100-fold excess of the 30-nucleotide RNA containing the stem-loop sequence. Control, 0.1 ng of a synthetic 77-nucleotide RNA containing the complete sequence of the U7 snRNA added to each sample as an internal standard for RNA recovery and hybridization efficiency.

FIG. 6

The stimulation of binding of the U7 snRNP by SLBP is distance dependent. (A) The sequence of the H2a-614 mutant substrates containing either 4- or 12-nucleotide insertions between the normal cleavage site and the HDE. (B) The radiolabeled substrates shown in panel A and indicated above each lane were incubated in nuclear extract under standard processing conditions, and RNA was analyzed as described above. (C) The anti-SLBP antibody was used to isolate processing complexes assembled on the pre-mRNA substrates, as described in Fig. 5C. Immunoprecipitation with no pre-mRNA added (lane 1) or in the presence of the substrates shown in panel A (lanes 2 to 4). (D) The anti-SLBP antibody was used to isolate processing complexes assembled on the H2a-614 substrate (lanes 2 and 3) in the control nuclear extract (NE) or in extract which had been heat inactivated at 50° for 15 min. Lane 1, no RNA was added to the control extract.