Length suppression in histone messenger RNA 3'-end maturation: processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA

Abstract

Efficient 3'-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem-loop and a histone downstream element (HDE) that base pairs with the U7 small ribonucleoprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Figure 1

Diagram of mouse histone H2A pre-mRNA and human U7 RNA mutants. Partial sequence of the mouse histone H2A pre-mRNA (4), indicating the wild-type cleavage site, the site of insertions, the HDE residues that base pair to the U7 RNA, and the residues altered in the base substitution mutants. All histone pre-mRNA substrates contain an additional 14 nt of vector sequence at the 5′ end. The human U7 sequence (5) is shown with the Sm site shaded. Partial sequences of the base substitution HDE and U7 RNA suppressor mutants (6) are shown with altered residues dipicted below the wild-type sequence. Residues involved in base pairing to the U7 RNA or the histone pre-mRNA are underlined with G·U base pairs indicated by dots.

Figure 2

In vitro and oocyte processing of histone pre-mRNA insertion mutants and HDE base substitution mutants. (A) Internally labeled insertion substrates were processed in HeLa cell nuclear extract (lanes 1–10) or injected into the GV of untreated Xenopus oocytes (lanes 11–20), of oocytes where the endogenous Xenopus U7 RNA was degraded and subsequently rescued by injection of in vitro-transcribed human U7 RNA (lanes 21–30), or oocytes where the endogenous Xenopus U7 RNA had been degraded (lanes 31–40). Note that lane 29 was underloaded in this particular experiment. In at least six different experiments, progressive decreases in processing efficiency were observed with longer insertions. (B) Suppression of substitution mutations in the HDE in Xenopus oocytes. Wild-type histone pre-mRNA was injected into untreated oocytes (lane 1), oocytes depleted of U7 RNA (lane 2), depleted oocytes rescued with U7wt RNA (lane 3), or depleted oocytes rescued with U7py→pu (lane 4). In lanes 5–7, the HDEpu-py substrate was injected into untreated oocytes, U7wt rescued oocytes, or U7py→py rescued oocytes. In lanes 8–10, the HDEgag→cuc mutant was injected into untreated, U7wt rescued, or U7cuc→gag rescued oocytes. In lanes 11–13, the HDEaaa→uuu mutant was injected into untreated, U7wt rescued, or U7 uuu→aaa rescued oocytes.

Figure 3

Processing of histone insertion mutant pre-mRNAs in the presence of length suppressor U7 RNAs. (A) Partial sequences of histone insertion substitution mutants and U7 RNA length suppressor mutants. The 5C insertion in the histone pre-mRNA is indicated by block letters while further base substitutions in this substrate are listed below. The point of insertion into U7 RNA is marked, with the inserted residues in blocked residues and the Sm site shaded. (B) In lanes 1–4, histone pre-mRNAs HDEpu→py, wild-type, 5C, and 10C were injected into untreated oocytes. The same histone pre-mRNAs were injected into depleted oocytes rescued with the following material. Lanes: 5–8, in vitro-transcribed U7wt RNA; 9–12, U75bp RNA; 13–16, U75nobp RNA; 17–20, U710 RNA; 21–24, not rescued.

Figure 4

Processing of 5C insertion/base substitution mutants in the presence of U75nobp. Lanes 1–6 contain in vitro processing of pre-mRNAs HDEpu→py, wild-type, 5C, 5C+2bp, 5C+3bp, and 5C+5bp, respectively. The same substrates were injected into the following oocytes. Lanes: 7–12, untreated Xenopus oocytes; 13–18, oocytes depleted of Xenopus U7 RNA and rescued with U7wt RNA; 19–24, depleted oocytes rescued with U75nobp RNA; 25–30, depleted oocytes.

Figure 5

Models of histone pre-mRNA processing complexes. The wild-type situation is compared with complexes formed on the 5C insertion substrate with the wild-type U7 snRNP, U75nobp snRNP, and the U75bp snRNP. Arrows show the sites of cleavage with levels indicated by arrow size. SLBP is shown as a striped oval, core Sm proteins bound to the Sm binding site (boxed) are different shades of gray, the two known U7 snRNP specific proteins are white ovals, and the hypermethylated cap on the 5′ end of U7 RNA is a solid circle. Base pairing between the U7 RNAs and the substrate HDE sequences is indicated. Insertions into the histone pre-mRNA and the U7 RNAs are illustrated as open and solid circles, respectively. Rigidification of the residues upstream of the HDE is diagrammed as involving protein–backbone contacts (see text).