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 cycleregulated mammalian histone premessenger RNAs (premRNAs) requires an upstream stem-loop and a histone downstream element (HDE) that base pairs with the U7 small ribonuclearprotein. 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.Cell cycle-regulated histone premessenger RNAs (premRNAs) are unusual RNA polymerase II transcripts because they do not contain introns and are not polyadenylylated (for review, see refs. 1 and 2). Instead, their 3Ј ends are formed by endonucleolytic cleavage. Two conserved elements on the histone pre-mRNA are required for efficient processing: a stem-loop structure found directly upstream of the site of cleavage and a purine-rich region located 9-17 nt (usually 11 Ϯ 1 nt, see ref.3) downstream of the cleavage site termed the histone downstream element (HDE) (Fig. 1). There are at least three trans-acting factors involved in histone mRNA maturation: a factor that binds the stem-loop element (7-11), an essential heat-labile factor whose role is not yet understood (12-14), and the low-abundance U7 small nuclear ribonucleoprotein (snRNP) (for reviews, see refs. 15 and 16), which contains at least two proteins of 50 kDa and 14 kDa (17-19) in addition to the core Sm proteins (20). Genetic suppression experiments established that the 5Ј end of the U7 RNA base pairs with the purines of the HDE during histone pre-mRNA processing (6, 21) (see Fig. 1); the ability to form an small nuclear RNA (snRNA)-pre-mRNA duplex, rather than the exact sequence of the complementary regions, is critical for processing.Previously, we reported the effects of systematically moving the HDE further downstream of the stem-loop in a mouse histone H2A pre-mRNA (22). The processed products generated in a HeLa cell in vitro system revealed that the site of 3Ј-end formation moves in concert with the movement of the HDE, remaining 11 Ϯ 1 nt upstream. Decreases in processing efficiency were also observed as the HDE was distanced from the stem-loop. These results suggested that the U7 snRNP directs cleavage at a fixed distance from its binding site (term...

Formation of the 3′ end of histone mRNA: Getting closer to the end

“…In some pre-mRNAs the stem-loop can be mutated without significantly affecting the efficiency of 3' end processing, whereas mutations within the HDE invariantly result in complete inhibition of the cleavage reaction (Mowry et al, 1989;Vasserot et al, 1989). Moving the HDE by as few as 5-6 nucleotides away from the stem-loop results in either complete (Georgiev and Birnstiel, 1985) or partial inhibition of 3' end processing (Scharl and Steitz, 1996;Scharl and Steitz, 1994). Thus, the spatial arrangement of the two processing signals in histone premRNAs is important for processing and suggested the existence of a communication between factors interacting with each of the two elements.…”

“…2). Blocking the U7 snRNA by a complementary oligonucleotide (Cotten et al, 1991) or mutations within the HDE that reduce the base pair potential to U7 snRNA abolish 3' end processing (Bond et al, 1991;Scharl and Steitz, 1996;Schaufele et al, 1986). The negative effect of the HDE mutations can be restored by compensatory mutations within the U7 snRNA, demonstrating that the formation of an RNA hybrid rather than the exact sequence of each RNA element involved in base pairing is essential for processing (Bond et al, 1991;Scharl and Steitz, 1996;Schaufele et al, 1986).…”

“…The reduced processing efficiency and the shift of the cleavage site resulting from the separation of the bipartite processing element in mammalian histone pre-mRNA can be fully suppressed by using modified U7 snRNAs containing comparable insertions between the Sm site and the region complementary to the HDE (Scharl and Steitz, 1996). The extended U7 snRNA restores the original configuration of the processing complex by bringing the Sm site and the attached Sm core, including Lsm11, to the proximity of the stem-loop and normal cleavage site.…”

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

“…Characterization of the processing of histone pre-mRNA derived from injected DNA+ A: The S1 nuclease protection assay used to measure histone pre-mRNA processing is diagramed+ The 260-nt probe contains 43 nt that are not present in the H2a-614 histone pre-mRNA, but are derived from the vector pBluescript KS (zig-zag line)+ The unprocessed RNAs protect a 217-nt fragment regardless of their 39 end and the processed histone mRNAs protect a 183-nt fragment+ Because the probe is 39-end labeled, the ratio of the intensity of the two fragments is a measure of processing efficiency+ B: Frog oocytes were preincubated in buffer (lane 2) or with 200 mg/mL Actinomycin D (lane 3) for 1 h+ They were then injected with the histone H2a-614 gene and blue Dextran, and incubated in the continued presence of the inhibitor for 18 h+ RNA was prepared and then assayed for histone H2a-614 mRNA+ Lane 1 is marker pUC18 digested with MspI+ C: Oocytes were injected with the H2a-614 gene and blue Dextran, incubated for 18 h and then half of the oocytes were harvested (lane 1) and the other half were incubated for an additional 5 h in 200 mg/mL Actinomycin D (lane 2)+ A separate batch of oocytes was injected with either the H2a-614 gene (lane 3) or with the histone H2a-4G gene (lane 4) and incubated for 18 h+ The H2a-4G gene produces a transcript that cannot be processed because of the insertion of four Gs at the cleavage site )+ RNA was prepared from the oocytes and RNA from the equivalent of one oocyte analyzed by S1 nuclease mapping+ D: Frog oocytes were injected with the histone H2a-614 gene and the levels of processed and unprocessed histone mRNAs measured 18 h later (lane 1)+ In lane 2, the human U7 gene (Jacobs et al+, 1999) was coinjected into the nucleus with the H2a-614 gene+ In lane 3 the oocytes were injected with synthetic xSLBP1 mRNA 30 h prior to injection of the histone H2a-614 gene+ In lane 4, the oocytes were injected with the xSLBP1 mRNA 30 h prior to coinjection of the human U7 gene and the histone H2a-614 gene+ The processing efficiency (percent processing) quantified using a PhosphorImager is indicated below each lane+ xSLBP1 and U7 snRNP cooperate to process histone pre-mRNA in vivo Histone pre-mRNA processing in oocytes requires both xSLBP1 (Wang et al+, 1999) and the U7 snRNP (Scharl & Steitz, 1996) as well as other factors+ A critical step in vitro is assembly of a stable complex containing SLBP and U7 snRNA on the histone pre-mRNA , and efficient assembly of the complex requires both stable binding (high affinity) of SLBP to the stem-loop and base-pairing of U7 snRNP to the pre-mRNA )+ To determine whether xSLBP1 or U7 snRNP were limiting for processing in vivo we increased the concentration of U7 snRNA by expressing U7 snRNA from the human U7 snRNA gene (Jacobs et al+, 1999) and the concentrations of xSLBP1 by injecting synthetic xSLBP1 mRNA+ There are excess snRNP proteins present in the frog oocyte that will assemble U7 snRNA into functional U7 snRNP particles (Strub & Birnstiel, 1986)+ In this experiment we used a batch of oocytes that processed the histone H2a-614 mRNA relatively inefficiently (Fig+ 1D, lane 1)+ Overexpression of either U7 snRNA or xSLBP1 resulted in an increased efficiency of processing (Fig+ 1D, lanes 2 and 3)+ Overexpression of both xSLBP1 and U7 snRNA in the same oocytes resulted in a further increase in processing (Fig+ 1D, lane 4)+ Thus both U7 snRNP and xSLBP1 are present at suboptimal concentrations for processing the histone H2a-614 pre-mRNA expressed in the oocyte+ This result is consistent with the suggestion that these two factors cooperate in histone pre-mRNA processing …”

Evolutionary conservation of the U7 small nuclear ribonucleoprotein inDrosophila melanogaster

“…The minor U7 snRNP binds to the purine-rich histone downstream element (HDE; Schaufele et al 1986;Bond et al 1991) and thereby serves as a molecular ruler, positioning the yet unknown cleavage activity near the processing site (Scharl andSteitz 1994, 1996). U7 snRNA is 58-71 nucleotides long, depending on the species, and like spliceosomal snRNAs, it bears a trimethyl guanosine cap at its 5Ј end.…”