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Functional Importance of Conserved Nucleotides at the Histone RNA 39 Processing Site

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Functional Importance of Conserved Nucleotides at the Histone RNA 39 Processing Site RNA (1998), 4:246–256+ Cambridge University Press+ Printed in the USA+ Copyright © 1998 RNA Society+ Functional importance of conserved nucleotides at the histone RNA 39 processing site ANDRÉ FURGER,1 ANDRÉ SCHALLER, and DANIEL SCHÜMPERLI Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, 3012 Bern, Switzerland ABSTRACT Histone pre-mRNA 39 processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA9CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 39 processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP.A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 59 of the cleavage site in the order A. C . U . G. Moreover, a guanosine in the 39 position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614–2620]. Keywords: adenosine; cleavage site; 39 end formation; mutational analysis; U7 snRNP INTRODUCTION despite the fact that the U7 snRNP participates in his- tone RNA 39 processing+ However, in neither of these Metazoans generate their mRNAs from longer precur- systems has the active nuclease been identified+ sor mRNAs (pre-mRNAs) by two distinct types of cleav- Two conserved sequence elements on the pre-mRNA age reactions+ The bulk of mRNAs are polyadenylated substrate are important for the formation of mature his- and, here, the cleavage is tightly linked to poly(A) ad- tone mRNA 39 ends (Birnstiel & Schaufele, 1988)+ (1) A dition, although the two steps can be dissociated in vitro hairpin element upstream of the cleavage site is bound (reviewed in Wahle & Keller, 1992)+ In contrast, the fam- by a cognate hairpin binding factor (Mowry et al+, 1989; ily of replication-dependent histone mRNAs are gener- Vasserot et al+, 1989)+ To reflect the fact that this trans- ated by a single cleavage event (reviewed in Birnstiel & acting component has been cloned recently and hence Schaufele, 1988)+ Notwithstanding many dissimilarities its composition is known (Wang et al+, 1996; Martin in cis-acting sequence elements and trans-acting fac- et al+, 1997), we have renamed it hairpin binding pro- tors, both types of reactions are independent of cleav- tein (HBP; Martin et al+, 1997)+ (2) A purine-rich spacer able ATP and divalent cations (Moore & Sharp, 1985; element located several nucleotides downstream of the Gick et al+, 1986; Wittop Koning, 1993) and the resulting cleavage site interacts by base pairing with the 59 end products contain 39 hydroxyl and 59 phosphate groups of U7 RNA present in the U7 snRNP (Schaufele et al+, (Moore & Sharp, 1985; Streit et al+, 1993)+ The lack of a 1986; Bond et al+, 1991)+ requirement for divalent cations suggests that both re- In addition to these two elements, the nucleotides actions may be protein catalyzed (Steitz & Steitz, 1993), immediately following the hairpin and encompassing the processing site show considerable sequence con- Reprint requests to: Daniel Schümperli, Abteilung für Entwicklungs- servation (Fig+ 1)+ In vertebrate histone genes, the first biologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, , ; : + + + + six nucleotides following the hairpin fit the consensus 3012 Bern Switzerland e-mail daniel schuemperli@zoi unibe ch + , 1Present address: Institut für Allgemeine Mikrobiologie, Baltzer- ACCCAC The seventh nucleotide is more variable but strasse 4, 3012 Bern, Switzerland+ shows a preference for A in mammals and U in non- 246 Conserved sequence of histone RNA 39 processing site 247 ucts whose 59 ends map to several positions between the 39 end of the 59 fragment and the spacer element (Gick et al+, 1986; Streit et al+, 1993; Fig+ 2A)+ Because we wanted to introduce mutations near the cleavage site(s) generating the mature mRNA (i+e+, the 59 frag- ment), it was important to define precisely the site of this cleavage+ For the 12/12 RNA used in this study, we have previously compared the cleavage products generated from 59-and 39 end-labeled RNAs by coelectrophoresis with the products of chemical sequencing reactions (Streit et al+, 1993)+ Since then, we have become aware that, whereas this determination provided correct re- sults for the 39 fragments, it most likely did not for the 59 fragments+ The reason is that the aniline strand scis- sion used in chemical sequencing of RNA (Peattie, 1979) FIGURE 1. Distribution of nucleotides (in absolute numbers) in the only leads to complete b-elimination on the 39 side of the first seven positions following the RNA hairpin of vertebrate histone modified nucleoside+ The 59 fragment still carries rem- genes+ Sequences of mammalian histone genes found in the GenBank/EMBL sequence library were from mouse (23 genes), rat nants of the modified nucleoside and aniline ring at its (11), and man (25)+ Nonmammalian vertebrate sequences were from 39 end and therefore has an anomalous electrophoretic rainbow trout (5 genes), Tilapia nilotica (1), Xenopus laevis (26), mobility (C+ Leumann, pers+ comm+)+ This is in contrast chicken (26), and duck (5)+ Note that one of the nonmammalian entries ends with the sixth nucleotide+ The most frequent nucleotide to the piperidine strand scission used in chemical se- at each position is shown in enhanced tone+ The seventh nucleotide quencing of DNA that leads to complete b-elimination on is the least conserved and shows a slight preference for A in mam- both the 39 and 59 side (Maxam & Gilbert, 1981)+ malian and U in nonmammalian species+ Above the compilation of mammalian sequences, the sequence of the mouse H4-12 gene We therefore compared the histone-specific process- used in this work is shown with indices representing the positions ing products of 59 end-labeled 12/12 RNA by coelec- after the hairpin+ trophoresis with adenosine-specific enzymatic and chemical sequencing products, as well as with an al- mammalian vertebrates+ Most of the 59 processing prod- kaline mononucleotide ladder generated from the same + + ucts mapped so far terminate with ACCC or ACCCA RNA (Fig 2B) Ribonuclease U2 cleaves after aden- , + (Gick et al+, 1986; Scharl & Steitz, 1994)+ Although some osines leaving a 29 or 39 phosphate group Because of these conserved nucleotides may be important for histone RNA processing leaves a 39-terminal hy- , , the binding of HBP (Williams & Marzluff, 1995) and droxyl group (Wittop Koning 1993) its product— certain mutations in this region have been found to should the cleavage be after an adenosine—will differ , reduce processing efficiency (Georgiev & Birnstiel, 1985; by a single phosphate group causing it to migrate + + Spycher et al+, 1994), this sequence has not been an- approximately 0 5 nt more slowly Figure 2B shows alyzed in detail+ We therefore have generated and an- that histone-specific cleavage (lane 2) indeed pro- alyzed mutations of the ACCCA9CA sequence in a duces a major 59 cleavage product migrating slightly mouse histone H4 pre-mRNA+ Mutations in the last slower than the U2 product (lane 3) generated from , + +, + three of these nucleotides have strong effects on both cleavage after A5 i e after ACCCA A minor cleav- the efficiency and site of cleavage in vitro+ Our results age product ending two nucleotides further down- , + +, , + confirm previous findings indicating that cleavage oc- stream i e after ACCCACA is also observed The + , curs preferentially downstream of A residues (Scharl & chemical sequencing ladder (Fig 2B lane 4) is re- Steitz, 1994), but also reveal the relative preference for tarded by one to two nucleotides with respect to the cleavage at other nucleotides+ Preference for adeno- enzymatic one and the extent of this shift varies slightly + , sines at the cleavage site is a shared feature with the along the gel Therefore only the enzymatic se- cleavage involved in polyadenylation, but the order of quence ladder can be used to appropriately deter- + cleavage preferences for the other nucleotides is dif- mine the end of the 59 cleavage products Compared +, , ferent+ Finally, the nucleotide immediately following the to our previous study (Streit et al 1993) the two cleaved phosphodiester bond also has an influence on cleavage sites are therefore located two nucleotides + the cleavage specificity+ further downstream The new positions (shown in Fig+ 2A) are in agreement with results of Scharl and Steitz (1994), who showed that the in vitro process- RESULTS ing product of a mouse H2A pre-mRNA ended after the sequence ACCCA+ This new assignment of the Definition of the cleavage site cleavage
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