Binding of human SLBP on the 3'-UTR of histone precursor H4-12 mRNA induces structural rearrangements that enable U7 snRNA anchoring

Abstract

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3' end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem-loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds to the stem-loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3'-untranslated region (3'-UTR) of mouse histone pre-mRNAs H4-12, H1t and H2a-614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3'-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4-12 pre-mRNAs 3' end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.

Figure 1

Schematic representation of the 3′ end processing of replication-dependent histone pre-mRNAs. Cleavage of the RNA occurs at the red arrow between a highly conserved stem–loop structure and the HDE. Trans-acting factors are SLBP, which binds to the hairpin structure, U7 snRNP which anchors the pre-mRNAs by annealing to HDE and the cleavage complex including CPSF-73 which is believed to be the cleavage factor. U7 snRNP contains two U7-specific proteins named Lsm10 and Lsm11. ZFP100 bridges U7 to histone pre-mRNAs by interacting with SLBP and Lsm11. The sequences of the 3′-UTRs used in this work are indicated below the cartoon. Nucleotides shown in bold were added to optimize transcription reactions. Base-pairing of H4-12, H1t and H2a-614 with mouse U7 snRNA is shown at the bottom of the figure.

Figure 2

Establishment of an experiment-based 2D model of the 3′-UTR of histone H4-12 pre-mRNAs in free state and effect of SLBP binding. (A) Chemical and enzymatic structural probing. Probing was done in the presence (+) or absence (−) of recombinant SLBP. AH and T1 are ladders of the RNA under denaturing condition; Ctrl is the control without any probe. Probes were Pb²⁺, RNase T2, RNase T1 and RNase V1. Domains of the stem–loop structure, the processing site (*) and the spacer element are indicated on the right part of the gel. (B) 2D model of the 3′-UTR of histone H4-12 pre-mRNAs with probing data. The colour code for the probes is indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium, moderate). The spacer element is highlighted in yellow. (C) Changes in the probing signals on SLBP binding shown on the RNA folding. Protection by SLBP is shown in black (signal reduction) and cut/modification appearance and enhancement (signal enhancement) are shown in white; symbols for each probe are the same as in (B). The presumed SLBP binding domain, deduced from cleavage protections, is coloured in light yellow. Green arrows symbolize opening of stem IV on SLBP binding.

Figure 3

Electrophoretic Mobility Shift Assay using U7 snRNA, H4-12 pre-mRNAs transcripts, SLBP protein and competitors. The 5′-radiolabelled U7 snRNA was incubated with 0.01 μM H4-12 pre-mRNAs in the absence (lane 4) or presence (lane 5) of recombinant SLBP (10 μM). In order to assess the specificity of hybridization between U7 snRNA and H4-12 3′-UTR, competition experiments were performed using a DNA–oligonucleotide corresponding to HDE sequence both in the absence (lane 3) or presence (lane 7) of SLBP. The (U7: DNA–oligonucleotide) complex is clearly separated from U7 RNA (see the less exposed autoradiogram in the lower part of the figure). To demonstrate that the observed effects are a direct consequence of SLBP binding to the 3′-UTR, competition experiments were done by adding RNA-competitor stem–loops. Whereas the wild-type competitor stem–loop decreased the level of U7 transcripts annealed to the 3′-UTR (lane 9), the asterisk-mutated competitor stem–loop (*) that exhibits no homology with wild-type stem–loop has no effect (lane 11 versus lane 5, see experimental procedures for the sequences of the stem–loops).

Figure 4

2D model of the 3′-UTR of histone H1t pre-mRNAs in free state and effect of SLBP binding. (A) Chemical and enzymatic probing data. (B) 2D model of the RNA. (C) Changes in the probing profile induced by SLBP binding are shown on the RNA 2D model. Legend is the same as for Figure 2.

Figure 5

2D model of the 3′-UTR of histone H2a-614 pre-mRNAs in free state and effect of SLBP binding. (A) Chemical and enzymatic probing data. (B) 2D model of the RNA. (C) Changes in the probing profile induced by SLBP binding are shown on the RNA 2D model. Legend is the same as for Figure 2.