.=) 1991 Oxford University Press

Nucleic Acids Research, Vol. 19, No. 18 4891 -4894

Proposed secondary structure of eukaryotic U14 snRNA Gamila M.Shanab and E.Stuart Maxwell* Department of Biochemistry, Box 7622, North Carolina State University, Raleigh, NC 27695-7622, USA Received June 28, 1991; Accepted August 7, 1991

ABSTRACT U14 snRNA is a small nuclear RNA that plays a role in the processing of eukaryotic ribosomal RNA. We have investigated the folded structure of this snRNA species using comparative analysis of evolutionarily diverse U14 snRNA primary sequences coupled with nuclease digestion analysis of mouse U14 snRNA. Covariant nucleotide analysis of aligned mouse, rat, human, and yeast U14 snRNA primary sequences suggested a basic folding pattern in which the 5' and 3' termini of all U14 snRNAs were base-paired. Subsequent digestion of mouse U14 snRNA with mung bean (single-strandspecific), T2 (single-strand-preferential), and VI (double-strand-specific) nucleases defined the major and minor cleavage sites for each nuclease. This digestion data was then utilized in concert with the comparative sequence analysis of aligned U14 snRNA primary sequences to refine the secondary structure model suggested by computer-predicted folding. The proposed secondary structure of U14 snRNA is comprised of three major hairpin/helical regions which includes the helix of base-paired 5' and 3' termini. Strict and semiconservative covariation of specific base-pairs within two of the three major helices, as well as nucleotide changes that strengthen or extend basepaired regions, support this folded conformation as the evolutionarily conserved secondary structure for U14 snRNA.

synthesis results in the disruption of pre-rRNA processing (3). Consistent with this role in the maturation of rRNA precursors, U14 snRNA can intermolecularly base-pair with 18S rRNA (6). Mouse U14 snRNA is 87 nucleotides in length (6) and found in the nucleolus associated with the conserved nucleolar protein fibrillarin (Maxwell, unpublished results). Fibrillarin is an RNA binding protein common to those snRNAs which play a role in rRNA processing such as U3, U8, and U13 (7). U14 snRNA also possess conserved nucleotide box C and D sequences found in U3, U8, and U13 nucleolar snRNAs (7, 8). More recently, cloning of the mouse U14 snRNA genes has revealed the positioning of three U14 snRNA coding sequences within three different introns of the constitutively-expressed mouse cognate hsc7O heat shock gene (8). The location of the U14 snRNA genes on the coding strand of the hsc70 gene has suggested the possible synthesis of mouse U14 snRNA via an intron-processing pathway (8). U14 snRNA's base-pairing interactions with 18S rRNA, the binding of fibrillarin, and the possible processing of U14 snRNA transcripts from the hsc7O pre-mRNA precursor will almost certainly be governed by the folded structure of this snRNA species. Therefore, determination of U14 snRNA secondary stucture will be important step in understanding structure/function relationships. Towards this goal, we have examined the secondary structure of mouse U14 snRNA using evolutionary analysis of conserved U14 snRNA primary sequence/secondary structure in conjunction with nuclease digestion analysis. In this work, we propose a conserved secondary structure that is exhibited by all presently known U14 snRNA sequences.

INTRODUCTION Analysis of nuclear RNA biosynthetic pathways has clearly demonstrated the importance of small nuclear RNAs in the processing of both eukaryotic messenger and ribosomal RNA precursor transcripts. Specific subsets of snRNAs are essential for the splicing of pre-messenger RNA (1) as well as for the cleavage of rRNA primary transcripts to yield mature species of 18S, 5.8S, and 28S rRNAs (2, 3). One such nucleolar snRNA required for rRNA processing is snRNA U14. U14 snRNA is widely dispersed in eukaryotes and is found in such evolutionarily diverse organisms as fungi, amphibians, mammals, and plants (4, Maxwell, unpublished results). U14 snRNA is encoded by an essential gene in yeast (5) and repression of U14 snRNA *

To whom correspondence should be addressed

METHODS AND MATERIALS Materials

[r-32P]Adenosine triphosphate (3000 Ci/mmole) was purchased from New England Nuclear Corp. and mung bean, T2, and VI ribonucleases were purchased from Pharmacia. Methods Preparation and Radiolabeling of Mouse U14 snRNA. Full-length transcripts of mouse U14 snRNA were produced by in vitro transcription of plasmid pGS-D/16 (details of plasmid construction will be reported elsewhere). These in vitro transcripts were 87 nucleotides in length and corresponded to the full-length

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4892 Nucleic Acids Research, Vol. 19, No. 18

U14 snRNA primary sequence (intron 5) that was determined by RNA sequencing (6). The 5' terminal uridine nucleotide has been replaced by a guanosine residue to increase transcriptional efficiency. Gel-purified U14 snRNA transcripts were radiolabeled at the 5' terminus using 32P-ATP and then repurified on 10% polyacrylamide-7M urea gels as previously detailed (4).

Ribonculease Mapping of U14 snRNA Secondary Structure. Terminally-labeled U14 snRNA (between 20 and 50 ng) was heated at 65°C for 5 minutes in 3t1 of H20 and then allowed to cool slowly to 37°C when appropriate nuclease digestion buffers were added. This mixture was then further cooled to 23°C. Digestion with RNases T2 and cobra venom VI was accomplished in a buffer of 20 mM Tris-HCl (pH 7.6), 0. IM NaCl, and 0, 1, or 2mM MgCl2. Digestion with mung bean nuclease was carried out in 20 mM NaAc (pH 4.6), 0. IM NaCl, and 0, 1, or 2mM MgCl2. Total reaction volumes of 5y1 were incubated at 30°C for 10 minutes in the presence of empiracally determined concentrations of the respective nucleases (see figure legend). Digestion reactions were stopped by adding an equal volume of standard TBE gel loading buffer containing 2.5mM EDTA and 0.1% SDS. Terminated digestion reactions were then heated at 65°C for 3 minutes before loading onto RNA sequencing gels. ALkaline hydrolysis and RNase T1 digestion of labeled U14 snRNA for marker lanes was carried out as previously detailed (9). Control samples were treated identically except that during nuclease digestion the nucleases were omitted.

RESULTS

Initial assessment of U14 snRNA secondary structure was begun by alignment of all ten known U14 snRNA primary sequences (Figure 1). High sequence homology was observed throughout most of the molecule. The highly conserved regions included the 5' and 3' terminally-located, nucleolar snRNA-specific box C

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and D sequences (underlined). The region of least conservation occured between nucleotides 47 and 52 (mouse intron 5 sequence). Lack of sequence conservation in this portion of the molecule, as well as inclusion of the yeast-specific 36 nucleotide insertion at this site, suggested that the secondary structure of U14 snRNA in this region would vary among the different folded U14 snRNA transcripts. Subsequent to this alignment, computer analysis was carried out (program provided by James Brown and Norman Pace) to search for strictly covarying nucleotide pairs in an effort to define potentially conserved base-pairs within the secondary structure of all U14 snRNAs. One such base-pair was noted and is indicated in Figure 1. The covariant nucleotides located at the 5' and 3' ends of the U14 snRNA molecules indicated that the 5' and 3' termini of all U14 snRNAs were basepaired to form a terminal helix as previously suggested (8). In vitro transcripts of mouse U14 snRNA (intron 5) were then digested with single-strand- and double-strand-specific ribonucleases to define the major and minor cleavage sites for this specific U14 snRNA transcript (Figure 2). No effect was observed in the digestion pattern when MgCl2 concentration was

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Figure 1. Alignment of U14 snRNA Primary Sequences. The primary sequences of mouse, rat, and human U14 snRNAs encoded in introns 5, 6, and 8 of the respective cognate hsc70 genes (8), as well as yeast U14 snRNA (5), are aligned to give the best homology fit. Conserved, nucleolar snRNA-specific, nucleotide box C and D sequences are underlined. The two asterisks located in the 5' and 3' terminal regions of the molecule indicate the nucleotide pair which strictly covaries in all U14 snRNA sequences. Primary sequences are numbered with respect to the sequenced mouse U14 snRNA transcript (6) which is shown above the aligned DNA coding sequences.

Figure 2. Nuclease Probing of Mouse U14 snRNA Secondary Structure. 5'-Radiolabeled mouse Ul4 snRNA was digested with T2, mung bean, or VI ribonuclease as detailed in methods and the resultant fragments resolved on an 8% polyacrylamide RNA sequencing gel. Lane 1, U14 snRNA digested with RNase TI to establish a 'G' map. Cleavage sites at G residues are indicated at the side and numbering is from the 5' end (+1) to the 3' end (+87). Lane 2, alkaline hydrolysis ladder. Lanes 3, 6, and 9, U14 snRNA digested with RNase T2 (0.25 U/gl) in the presence of 0, 1, and 2 mM MgCl2, respectively. Lanes 4, 7, and 10, U14 snRNA digested with mung bean RNase (0.5 U/l) in the presence of 0, 1, and 2 mM MgCl2, respectively. Lanes 5, 8, and 11, U14 snRNA digested with RNase VI (0.07 U/jl) in the presence of 0, 1, and 2 mM MgCl2, respectively. Lane 12, radiolabeled U14 snRNA control incubated in the absence of any added ribonuclease.

Nucleic Acids Research, Vol. 19, No. 18 4893 varied. Coincident with the digestion experiments, the Zuker RNA folding program (10) was utilized to predict the lowest free energy secondary structure for this mouse U14 snRNA sequence under the constraint that the 5' and 3' ends of the molecule be base-paired (Figure 3A). This computer-predicted secondary structure was then refined using the collected nuclease digestion data (Figure 3B). The nuclease cleavage sites observed in Figure 2 are indicated on both the computer-predicted and refined U14 snRNA secondary structures. Only those cleavage sites seen in Figure 2 are indicated on each of the presented U14 snRNA structures. Additional digestion experiments demonstrated the cleavage of both the 5' and 3' base-paired termini by the doublestrand-specific nuclease VI (data not shown). Refinement of the

computer-predicted secondary structure was not accomplished with strict adherence to the observed nuclease digestion sites from Figure 2, but was completed by the simultaneous folding and readjustment of these folded structures for all 10 U14 snRNA primary sequences given in Figure 1. The similar folding of the remaining U14 snRNA primary sequences into a consensus secondary structure is shown in Figure 4. Each folded U14 snRNA sequence consists of three major helical regions defined as helices I, II, and Ill [designated on the folded mouse U14 snRNA structure (intron 5)]. Helices I and II are more highly conserved than helix mII. Helix Im is that portion of the U14 snRNA primary sequence which is least conserved and contains the yeast-specific 36 nucleotide insertion.

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Proposed secondary structure of eukaryotic U14 snRNA.

U14 snRNA is a small nuclear RNA that plays a role in the processing of eukaryotic ribosomal RNA. We have investigated the folded structure of this sn...
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