molecular analysis of dicot and monocot small nuclear rna

The Plant Cell, Vol. 1, 633-643, June 1989 O 1989 American Society of Plant Physiologists

Molecular Analysis of Dicot and Monocot Small Nuclear RNA Populations

Daniel 6. Egeland, Ann Pizanis Sturtevant, and Mary A. Schuler’ Departments of Plant Biology and Biochemistry, University of Illinois, Urbana, lllinois 61 801

Oligonucleotides directed against conserved small nuclear RNA (snRNA) sequences have been used to identify the individual U1, U2, U4, U5, and U6 snRNAs in dicot and monocot nuclei. The plant snRNA populations are significantly more heterogeneous than the mammalian or Saccharomyces cerevisiae snRNA populations. U6 snRNA exists as a single species of similar size in monocot and dicot nuclei. The abundance and molecular weights of the U1, U2, U4, and U5 snRNAs expressed in monocot and dicot nuclei are significantly different. Whereas most dicot nuclei contain one or two predominant forms of U2 snRNA and a small number of U4 snRNAs, monocot nuclei contain multiple forms of U2 snRNA ranging from 208 to 260 nucleotides and multiple forms of U4 snRNA from 159 to 176 nucleotides. Multiple forms of U1 and U5 snRNA exist in both plant groups. All prominent size variants of U1, U2, U4, and U5 snRNA identified in monocot nuclei can be immunoprecipitated with anti-trimethylguanosine antibody. We conclude that the sizes and number of snRNA molecules involved in intron excision differ considerably in dicot and monocot nuclei. In wheat nuclei, we have identified an additional U1-like RNA that is differentially expressed during development.

INTRODUCTION

Nuclear genes in all eukaryotes are interrupted by one or more introns that must be excised from the initial transcript to produce mature, functional mRNA products. Although all higher organisms contain genes with introns, the mech- anisms of intron excision and the sequences that specify the process have been elucidated only in a small number of yeast and vertebrate cell types. Sequence comparisons in a variety of lower eukaryotes and metazoa have indicated that three sequence elements in each intron are necessary for the molecular ligation of exons. In metazoa, the first of these elements, the 5‘ intron boundary, is specified by a relatively conserved sequence of nine nucleotides invariably containing a GT dinucleotide at the exon/intron junction. A second element, found at the 3’ end of the intron, is an AG dinucleotide preceded by an 11 to 14 nucleotide pyrimidine tract (Mount, 1982). The third element critical for intron splicing in yeast and metazoa is a conserved sequence located between 20 and 50 nucleotides in front of the 3‘ splice junction (Langford and Gall- witz, 1983; Pikielny, Teem, and Rosbash, 1983; Keller and Noon, 1984, 1985). Although the degree of conservation in these three sequences varies significantly in metazoan and yeast introns, in vivo and in vitro RNA processing experiments have implicated all three of these conserved

’ To whom correspondence should be addressed. Current ad- dress: Department of Plant Biology, University of Illinois, 289 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61 801.

elements in the intron excision process (Padgett et al., 1986).

The complex set of events involved in pre-mRNA splicing is carried out by a large RNA-protein complex in which the precursor RNA is associated with a variety of small, nuclear RNAs (snRNAs) and nuclear proteins. Individual small nuclear ribonucleoprotein particles (snRNPs) within this complex are required for recognition of the three conserved intron elements. The U1 snRNP interacts directly with the 5’ splice site (Mount et ai., 1983; Black, Chabot, and Steitz, 1985; Zhuang and Weiner, 1986), and the U2 snRNP interacts with the conserved branch site within the intron (Black, Chabot, and Steitz, 1985; Krainer and Man- iatis, 1985; Bindereif and Green, 1987). The U5 snRNP particle has been implicated in recognition of the 3’ splice junction (Chabot et al., 1985). Although its function remains unclear, an additional snRNP, the U4-U6 snRNP, associ- ates with the other snRNPs in active splicing complexes (Berget and Robberson, 1986; Black and Steitz, 1986; Grabowski and Sharp, 1986; Bindereif and Green, 1987).

In contrast to the vast literature documenting the importance of snRNAs and snRNPs in mammalian and yeast intron excision, few efforts have carefully evaluated the nuclear components involved in plant intron excision. All plant introns have the GT-AG dinucleotide border elements found in mammalian and yeast introns (Mount, 1982; Teem et al., 1984; Brown, 1986; Brown, Feix, and Frendeway, 1986), but, in contrast to yeast and mammalian introns,

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634 The Plant Cell

plant introns exhibit a very high degree of sequence variability at the intron boundaries (Brown, 1986; Brown, Feix, and Frendeway, 1986; Hanley and Schuler, 1988). Exten- sive differences occur on the proximal side of the 3' splice junction in an area potentially interacting with the U5 snRNP particle. In this region of plant introns, purine-rich regions frequently replace the prominent pyrimidine-rich stretch preceding the 3'-terminal -AG found in mammalian introns (Hanley and Schuler, 1988). The purine- or pyrimidine-richness of this region has been used to divide plant introns into a purine-rich class that contains features of the highly conserved Saccharomyces cerevisiae introns and a pyrimidine-rich class that contains the sequence variations characteristic of mammalian introns (Hanley and Schuler, 1988). The proportion of purine and pyrimidine- rich introns varies significantly in monocots and dicots, and suggests that different snRNA or snRNP recognition factors exist in these two types of nuclei (Hanley and Schuler, 1988).

Small nuclear RNAs, the integral components of mammalian and yeast pre-mRNA splicing machineries, have not been well characterized in plant cells because the abundance of snRNAs in plant nuclei is 100-fold lower than in animal nuclei (1 04/nucleus versus 1 O'/nucleus) (Busch et al., 1982). Four of the plant snRNAs that have been characterized (Ul, U2, U4, U5) contain methylated cap structures [2,2,7-trimethylguanosine (m,G)] (Krol et al., 1983; Kiss, Toth, and Solymosy, 1985; Kiss et al., 1988). Although a limited amount of sequence information is available for the dicot snRNAs in pea (U1 , U2, U5; Krol et al., 1983), bean (U1 ; Van Santen and Spritz, 1987), broadbean (U2, U3, U4, U6; Kiss, Toth, and Solymosy, 1985; Kiss, Antal, and Solymosy, 1987a, 1987b; Kiss et al., 1988), soybean (U1 ; Van Santen, Swain, and Spritz, 1988), and Arabidopsis (Ul, U2; A.P. Sturtevant and R.E. Zielin- ski, in preparation; Vankan and Filipowicz, 1988), only one type of monocot snRNA (wheat U2) has been sequenced (Skuzeski and Jendrisak, 1985).

Throughout these studies, it has become clear that dicot snRNAs are more heterogeneous than their mammalian counterparts, which exist as unique species in most vertebrates (Busch et al., 1982). One-dimensional gels have indicated that dicot nuclei derived from pea, broadbean, tomato, tobacco, and cucumber contain discrete U2 and U6 snRNAs and multiple forms of U1 and U5 snRNA (Krol et al., 1983; Kiss, Toth, and Solymosy, 1985). On two- dimensional analytical gels that resolve 30 major pea snRNA species, the dicot U2 and U6 snRNAs fractionate as discrete species (Tollervey, 1987). Apart from one report (Skuzeski and Jendrisak, 1985) demonstrating that wheat nuclei contain a minimum of three U2 snRNA variants, none of the monocot snRNAs has been directly identified.

In this paper, we utilize a series of oligodeoxyribonucleotide probes to define snRNA diversity in dicot and monocot plants. We demonstrate that, in contrast to dicot

snRNA populations, which contain a few prominent variants of U2 and U4 snRNA, multiple forms of U2 and U4 snRNA exist in monocot nuclei. Multiple U1 and U5 snRNA species are expressed in both types of nuclei. In contrast, U6 snRNA is expressed as a single species of similar size in monocots and dicots. lmmunoprecipitation with anti- m3G antibodies has been used to demonstrate that all of the snRNA variants contain a trimethylguanosine cap. In addition, we have examined the expression of the heterogeneous monocot snRNA population in mature and dividing wheat nuclei.

RESULTS

High-Resolution Analysis of Pea and Wheat snRNAs

Because of the multitude of small RNA species in plant nuclei, the U1, U2, U4, U5, and U6 snRNA populations have been identified using the oligonucleotide probes defined in Table 1. The U1, U2, U4, and U6 probes are complementary to regions of the snRNAs conserved in a wide variety of organisms. The U1 oligonucleotide is complementary to the first 10 nucleotides that are present in all characterized U1 snRNAs (Siliciano, Jones, and Guthrie, 1987; Van Santen and Spritz, 1987; Reddy, 1988; Van Santen, Swain, and Spritz, 1988) and known to interact with the 5' splice site (Mount et al., 1983; Black, Chabot, and Steitz, 1985; Zhuang and Weiner, 1986). The U2 oligonucleotide is complementary to nucleotides 28 to 42 in U2 snRNA, a region absolutely conserved in mammals, S. cerevisiae, broadbean, and Arabidopsis (Ares, 1986; Kiss, Antal, and Solymosy, 1987b; Reddy, 1988; Vankan and Filipowicz, 1988). U5 snRNA is poorly conserved between vertebrates and plants (Krol et al., 1983; Reddy, 1988). Hence, the U5 oligonucleotide was designed to complement four of the pea U5 snRNAs in hairpin-loop I, the only conserved region in the vertebrate and plant U5 snRNA sequences (Krol et al., 1983). Because plant U4 and U6 snRNA sequences have only recently become available (Kiss, Antal, and Solymosy, 1987a; Kiss et al., 1988), the oligonucleotide probes used to detect these snRNAs were designed to complement regions conserved in the vertebrate and S. cerevisiae U4 and U6 snRNAs (Brow and Guthrie, 1988; Reddy, 1988). Comparison of these probes with the recently published sequences (Kiss, Antal, and Solymosy, 1987a; Kiss et al., 1988) indicates that the U4 and U6 oligonucleotides are complementary to 15/16 and 13/14 positions in the broadbean U4 and U6 snRNAs, respectively. The hybridization and wash conditions are low enough to permit the following numbers of base mismatches: U1, 2 to 3/13; U2, 3 to 4/15; U4, >3/ 16; U5, >3/13; U6, 2 to 3/14.

In preliminary studies on low-resolution gels, hybridization of each probe with dicot (pea) and monocot (wheat)

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Table 1. Oligodeoxyribonucleotide Probes

Oligonucleotide Complementary Nucleotides Length Oligo Sequence

1-13 28-42

53-68

35-47

38-51

13 15

16

5’-GCCAGGTAAGTAT-3’ 5’4AGATACTACACTTG-3’

cc l-r

5‘-TTTCAA AGCAATAA-3’

13 5 ' -TAGTAAAAGGCGA-3 ’ A

T 14 5’-TC TCTCTGTATTG-3’

U1-3‘AWa 135-1 52 18 5‘-GCGCGAACGCAGICCCCC-3’

On the right are the oligonucleotide sequences complementary to the specific snRNAs shown at the left. The positions of the complementary nucleotides in each snRNA are designated in the second column. The length of each oligonucleotide is shown in the third column. The snRNA sequences needed for this compilation were derived from a composite of sequences for U1 and U4 (Reddy, 1988), U6 (Brow and Guthrie, 1988; Reddy, 1988), U2 (Kiss, Antal, and Solymosy, 1987b; Reddy, 1988; Vankan and Filipowicz, 1988), and U5 (Krol et al., 1983).

nuclear RNA preparations revealed distinct differences between the pea and wheat snRNA profiles primarily in the patterns for the U1, U2, and U4 snRNAs. The U1 oligonucleotide hybridized with broad bands centered at 163 nucleotides (nt) and 170 nt, respectively. In contrast, the U4 oligonucleotide reacted with a discrete pea U4 snRNA (160 nt) and a diffuse set of wheat snRNAs between 159 and 168 nucleotides. As shown in Figure 1, on high-resolution sequencing gels, the pea and wheat U1 snRNAs can be separated into four species that are 160 to 167 nt and 168 to 185 nt, respectively. In wheat, an additional high molecular weight RNA (284 nt) cross-hybridizes strongly with the 5’ U1 oligonucleotide probe. This RNA will be discussed later. In pea, only one form of U4 snRNA (1 60 nt) can be resolved on these gels. In contrast, wheat U4 snRNAs, which were already heterogeneous on low-resolution gels, produce such a diffuse signal on the high resolution gels that it is difficult to define the exact number of U4 snRNAs in wheat. The apparent lengths of the U5 and U6 snRNAs were nearly identical in both plants (1 25 to 133 nt and 1 O1 to 104 nt, respectively). At high resolution, U5 snRNAs resolve as a broad band containing roughly three to four variants of equal intensity. Both types of plant nuclei contain a single U6 snRNA of similar size (101 to 104 nt).

More extensive differences between the dicot and monocot snRNA profiles are apparent in the U2 snRNA populations. As previously noted (Krol et al., 1983; Tollervey, 1987), pea nuclei contain one major (209 nt) form of U2 snRNA. In contrast, wheat nuclei contain multiple U2 snRNAs ranging from 208 to 260 nucleotides in length (Figure 1). On the high-resolution sequencing gels, we have resolved the wheat U2 snRNA population into seven species. Five major U2 snRNAs migrate with apparent

molecular weights between 208 and 239 nucleotides. Two less prominent U2 snRNAs migrate at 252 and 260 nucleotides. Under these conditions, the pea U2 snRNA is resolved into one major 209-nt species containing more than 95% of the U2 snRNA and a minor 205-nt species. Prolonged exposure of this RNA gel blot reveals severa1 small RNAs that react with the U2 oligonucleotide. The variability in these signals suggests that these RNAs represent U2 snRNA degradation products and not authentic U2 RNAs cross-reacting at low efficiency.

Comparison of Monocot and Dicot snRNAs

To determine whether these heterogeneous populations occur in other monocot and dicot nuclei, other plant RNA preparations were hybridized with the oligonucleotide probes shown in Table 1. The dicots used in this evaluation include pea, tobacco, Arabidopsis thaliana, and alfalfa. The monocots used include wheat, maize, sorghum, and barley. RNA gel blot analysis (Figure 2) indicates that the differences seen in the pea and wheat snRNA populations are characteristic of other dicot and monocot plants. This analysis also highlights the wide range of snRNA variants that are expressed within dicot species and within monocot species. In all of the monocots examined, there are multiple U1, U2, U4, and U5 species that fall within the size range defined for each of the wheat snRNAs. The dicot plants contain multiple U1 snRNAs (1 60 to 169 nt) that are consistently smaller than the monocot U1 snRNAs (1 68 to 185 nt). Dicot and monocot nuclei have multiple U5 sn- RNAs in the same size range. Whereas a small number of U4 snRNAs (about 160 nt) are present in dicot nuclei, multiple U4 snRNAs (160 to 176 nt) are expressed in

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U2Ul

U2

I1— -284

2097(205^

/239^230—224-^220^208

167= Jft160-W

-120(55)

I85168

-120(55)

' ii

U5(+U2)U6

133129

o ,s I •• of =€ CM

X "1Figure 1. High-Resolution Analysis of Pea and Wheat snRNAs.Pea and wheat nuclear RNAs were electrophoresed on 35-cm 10% acrylamide, 8.3 M urea denaturing gels. The resulting RNA gel blotwas sequentially hybridized with the 32P-labeled oligonucleotide probes shown in Table 1. The hybridization probe used is designated atthe top of each autoradiograph. (Some residual U2 snRNA is present on the blot hybridized with the U5 oligonucleotide.) Apparentmolecular weights calculated for the snRNAs are shown at the right and left of each autoradiograph. In each panel: Lane 1, 7.8 ^g of peanuclear RNA; lane 2, 8.2 ^g of wheat nuclear RNA; lane 3, 32P end-labeled 5S rRNA and tRNA standards or 32P end-labeled, denaturedpBR322 DNA. The first U2 panel, which is a prolonged exposure of the second panel, reveals several small degraded RNAs cross-reactingwith the U2 oligonucleotide.

monocot nuclei. (Barley snRNAs cross-react poorly withthe U4 oligonucleotide either because of their low abun-dance or their high degree of sequence divergence.) Alldicots tested contain one or two prominent 112 snRNAs.In alfalfa, two U2 snRNA variants of nearly identical sizefractionate as a broad band. Monocot nuclei contain aminimum of four U2 snRNA size variants. In dicots, themolecular weights of the U2 snRNAs (209 to 225 nt), whichvary more extensively than any other dicot snRNA, em-phasize the extreme size variations between dicot species.In contrast to the variations in the U1, U2, U4, and U5snRNA populations, all plant nuclei contain a single U6snRNA. Table 2 summarizes the molecular weight rangesfor plant snRNAs and compares them with mammalianand yeast snRNAs.

In our RNA gel blot analysis (Figure 2), the relative signalstrength obtained with the U1, U2, and U4 oligonucleotideprobes (relative to the U6 oligonucleotide) varied fromspecies to species, even though the oligonucleotides com-plement highly conserved regions in the snRNAs and lowstringency conditions were used for the hybridizations. Forexample, the pea and Arabidopsis RNA preparations con-tain proportionally more U2 snRNA than U1 or U4 snRNA.Tobacco and maize contain nearly equal proportions of U1and U2 snRNAs and significantly lower amounts of U4snRNA. Wheat and barley contain almost undetectablelevels of U4 snRNA. The most plausible explanation forthese variations is that the relative proportions of U1, U2,

and U4 snRNA fluctuate significantly in plant nuclei. Alter-nately, some of the U1 and U4 snRNA populations havediverged within the highly conserved regions representedby the oligonucleotide probes. Decreasing hybridizationstringency does not alter the relative proportions of thesnRNAs detected by RNA gel blot analysis (data notshown). In view of this and the high degree of conservationin these sequences (Van Santen and Spritz, 1987; VanSanten, Swain, and Spritz, 1988; Kiss et al., 1988; Reddy,1988), it is unlikely that entire groups of U1 and U4 snRNAshave diverged from the consensus sequences. We con-clude that the number of snRNA isoforms and the poolsizes of individual snRNAs are regulated independently inplant nuclei.

Immunoprecipitation of snRNA Variants

To determine whether the multiple monocot snRNA var-iants retained the 2,2,7-trimethylguanosine (m3G) capstructure characteristic of small nuclear RNAs, the wheatnuclear RNAs were immunoprecipitated with anti-m3G an-tibody, as described by Luhrmann et al. (1982). The im-munoprecipitated snRNAs were evaluated by direct RNAgel blot analysis and by 3' end-labeling with 32P-pCp andT4 RNA ligase (England, Bruce, and Uhlenbeck, 1980), asshown in Figure 3. The latter procedure defines the totalpopulation of immunoprecipitated RNAs and measures the

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selectivity of the immunoprecipitation reaction. The m3G-immunoreactive RNAs precipitated by this procedure in-clude all of the prominent U2, U1, U4, and U5 snRNAgroups previously discussed (Figure 3A). U6 snRNA, whichlacks a trimethylguanosine cap (Bringmann et al., 1983),is present in minor amounts presumably due to its base-paired association with U4 snRNA (Bringmann et al., 1984;

Table 2. Comparison of Plant, Animal, and Yeast snRNAs

<n

I 8 . .

I I I I I 1| S<s> to

225-

209-

169-160-

-260

-208U2

-I85-I68 Ul

—176

-I60 U4

I28-

IOI-

-I37-I26 U5

-I04 U6-IOI

Figure 2. RNA Gel Blot Analysis of Monocot and Dicot snRNAs.Dicot and monocot RNAs were electrophoresed on high-resolu-tion 10% acrylamide, 8.3 M urea denaturing gels, blotted ontoGene-Screen, and sequentially analyzed using the oligonucleotideprobes shown in Table 1. The identities of the hybridizing oligo-nucleotides are indicated at the right of each autoradiograph.Dicot RNA samples include: Lane 1, A thaliana; lane 2, tobacco;lane 3, alfalfa; lane 4, pea. Monocot RNA samples include: Lane5, wheat; lane 6, maize; lane 7, sorghum; lane 8, barley. Lanes 1to 3 and 6 to 8 contain 20 ng of total RNA; lanes 4 and 5 contain7.8 Mg of pea and 10.8 fig wheat nuclear RNA. Note that thedistance between the autoradiographs in the composite blot doesnot reflect the spacing of these snRNAs on acrylamide gels.

snRNAU2U1U4U5U6

Dicots209-225160-169160-166125-129101

Monocots208-260168-185160-176128-137101-104

Human Cells187164145116106

S. cerevisiae1175568159179/214112

The apparent sizes of the most abundant dicot and monocotsnRNAs taken from Figures 1 and 2 are shown in the second andthird columns. The molecular weights for human and S. cerevisiaesnRNA populations, derived from gel and sequence analyses, areshown in the fourth and fifth columns (Guthrie and Patterson,1988; Reddy, 1988).

Hashimoto and Steitz, 1984). Additional minor snRNAs, ofundetermined structure, are also immunoprecipitated withthe anti-m3G antiserum. RNA gel blot analysis (Figures 3Cto 3F) definitively demonstrates that most of the U1, U2,and U4 snRNA variants detected with the oligonucleotideprobes are immunoprecipitated with the anti-m3G anti-serum. The signals for the U2 snRNA population obtainedon RNA gel blots and by 3' end analysis differ slightly dueto variations in the efficiency of 3' end-labeling. Becausethe 3' end-labeled U1 and U4 snRNAs overlap in molecularweight (Figure 3A), RNA gel blot analysis (Figures 3D to3F) was used to define the trimethylguanosine-capped U1and U4 snRNA variants. The main sets of U1 snRNAs (168to 185 nt) and U4 snRNAs (156 to 163 nt), describedpreviously, were immunoprecipitated with anti-m3G anti-body. In addition, three low-abundance capped U4 snRNAvariants at 182 nt, 189 nt, and 198 nt were detected(Figure 3F).

Developmental Expression of a U1-like snRNA

In our earlier analyses (Figures 1 and 3D), an additional284-nt RNA cross-reacted with the oligonucleotide probecomplementary to the 5' end of U1 snRNA. Because thisRNA is substantially larger than the plant U1 snRNA se-quences previously characterized (Kiss, Toth, and Soly-mosy, 1985; Van Santen and Spritz, 1987; Van Santen,Swain, and Spritz, 1988), we have hybridized the anti-m3G-precipitated RNAs with an oligonucleotide (U1-3')directed against the 3' end of U1 snRNA (Figure 3E).Figures 3D and 3E clearly indicate that, although it cannotbe immunoprecipitated with anti-m3G antibody, the 284-ntRNA hybridizes with oligonucleotides directed against bothends of U1 snRNA. In fact, the 284-nt RNA hybridizes aswell or better than the lower molecular weight U1 snRNAvariants. Collectively, these results suggest that the 284-nt RNA potentially represents a U1-like RNA lacking atrimethylguanosine cap structure.

To determine whether the multiple wheat snRNA var-

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638 The Plant Cell

iants were differentially expressed during development,nuclear RNAs were isolated from immature, differentiating,and fully differentiated sections of wheat leaves. The RNApopulations were evaluated by 3' end-labeling and by RNAgel blot analysis. Terminal labeling with cytidine 3',5'-bisphosphate indicates that the small RNA populationsvary in mature and immature, dividing cells (data notshown). As shown in Figure 4, RNA gel blot analysis withthe U1 oligonucleotide demonstrates that the main set ofU1 snRNAs (168 to 185 nt) is expressed at equal levels inall stages of leaf development. Surprisingly, the 284-nt

RNA detected with the 5' and 3' U1 oligonucleotide probesis differentially expressed in these three developmentalstages. In the more differentiated distal sections, the 284-ntRNA is abundant, compared with the low molecular weightU1 snRNAs. The maturing cells in the medial section ofthe leaf contain less of the 284-nt species, and dividingcells in the basal section are devoid of this RNA. Similaranalysis with the other snRNA probes indicated that theU2, LI4, U5, and U6 snRNA populations do not changedramatically at any stage in leaf differentiation (data notshown).

C. uz D. ui E. ui-3' F. U4

" »I?11

Figure 3. Immunoprecipitation of Wheat snRNAs with Anti-m3G Antibodies.

(A) Wheat nuclear RNAs were immunoprecipitated with anti-m3G antibody as described by Luhrmann et al. (1982); 5-hr exposure.(B) Wheat nuclear RNAs were immunoprecipitated with anti-m3G antibody as described by Luhrmann et al. (1982); 14-hr exposure.(A) and (B) The immunoreactive RNAs were 3' end-labeled using 32P-cytidine bisphosphate and T4 RNA ligase (England, Bruce, andUhlenbeck, 1980), electrophoresed on high-resolution denaturing gels, and autoradiographed. Lane 1,32P end-labeled, denatured pBR322DNA standard; lane 2, 3.3 x 10" cpm end-labeled total nuclear RNA; lane 3, 6.9 x 10" cpm end-labeled anti-m3G immunoprecipitatednuclear RNA.(C) Wheat nuclear RNAs were immunoprecipitated with anti-m3G antibody, electrophoresed on high-resolution denaturing gels, andsubjected to RNA gel blot analysis using the U2 oligonucleotide probe.(D) Wheat nuclear RNAs were immunoprecipitated with anti-m3G antibody, electrophoresed on high-resolution denaturing gels, andsubjected to RNA gel blot analysis using the U1 oligonucleotide probe.(E) Wheat nuclear RNAs were immunoprecipitated with anti-m3G antibody, electrophoresed on high-resolution denaturing gels, andsubjected to RNA gel blot analysis using the U1-3'A oligonucleotide probe.(F) Wheat nuclear RNAs were immunoprecipitated with anti-m3G antibody, electrophoresed on high-resolution denaturing gels, andsubjected to RNA gel blot analysis using the U4 oligonucleotide probe.(C) to (F) Lane 1,32P denatured pBR322 DNA; lane 2, 5.4 /ig of wheat nuclear RNA; lane 3, anti-maG immunoprecipitated wheat nuclearRNA. Sizes for the molecular weight standards are shown on the left; snRNAs are identified on the right of each panel.

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B°- _•5 .2 o2 -o M.2 « oO 2 ffi

Cs) <M<r o:CD ma a

284- — -298

-220

-5.8 S

-154

-5S

Figure 4. Differential Expression of a 111-like snRNA in Wheat.Twenty micrograms of RNA isolated from the distal (0 cm to 2cm), medial (2 cm to 4 cm), and basal (4 cm to 6 cm) sections ofetiolated wheat leaves were electrophoresed on high-resolution10% acrylamide, 8.3 M urea gels and analyzed by RNA gel blotanalysis as described previously.(A) Ethidium bromide stained gel. Lane 1, 20 //g of wheat RNAfrom basal sections; lane 2, 20 fig of wheat RNA from medialsections; lane 3, 20 ng of wheat from distal sections.(B) RNA gel blot hybridization using the U1 oligonucleotide probecomplementary to nucleotides 1 to 13 in U1 snRNA. Lane 1, 20ng of wheat RNA from distal sections; lane 2, 20 ^9 of wheatRNA from medial sections; lane 3, 20 ^g of wheat RNA from basalsections; lanes 4 and 5, 32P end-labeled, denatured pBR322 DNAstandards. Molecular weights for the denatured pBR322 DNAstandards are shown at the right.

DISCUSSION

In spite of the intrinsic importance of snRNAs and snRNPsin intron excision, the snRNAs present in plant nuclei havenot been well characterized. Nearly all of the plant snRNAsthat have been characterized (Krol et al., 1983; Kiss, Toth,and Solymosy, 1985; Kiss, Antal, and Solymosy, 1987a,1987b; Kiss et al., 1988) are derived from dicot nuclei.None of the monocot snRNA populations has been ana-lyzed, even though several studies have indicated thatmonocot introns are ineffectively excised in dicot nuclei

(Keith and Chua, 1986). Using a series of oligonucleotideprobes complementary to regions conserved in mamma-lian, yeast, and plant snRNAs, we have analyzed thesnRNA components of monocot and dicot nuclei. The U1oligonucleotide complements the first 10 nucleotides of U1snRNA that are involved in base pairing with the 5' splicesite (Mount et al., 1983; Black, Chabot, and Steitz, 1985;Zhuang and Weiner, 1986). The U2 oligonucleotide com-plements nucleotides 28 to 42 in U2 snRNA that arerequired for interaction with the branch site sequence(Black, Chabot, and Steitz, 1985; Bindereif and Green,1987). The U4, U5, and 116 oligonucleotides are comple-mentary to internal sequences conserved in a wide varietyof organisms (Krol et al., 1983; Kiss, Antal, and Solymosy,1987a; Kiss et al., 1988; Reddy, 1988).

These probes, which effectively evaluate size hetero-geneities in snRNA populations, reveal that the patternsof snRNAs are surprisingly different in monocot and dicotnuclei and within each of these plant groupings. Becausethe probes cannot discriminate between closely relatedsnRNA variants of the same size, our results provideminimal estimates for the number of snRNA variants ac-tually expressed in plant nuclei. With this caveat, three tofive forms of U1 snRNA are abundantly expressed inmonocot and dicot nuclei. Complementarity with the 5' U1oligonucleotide, under conditions that permit 2 to 3/13base mismatches, demonstrates that each U1 snRNAvariant contains 5' nucleotides capable of interacting with5' splice sequences in introns (Mount et al., 1983; Black,Chabot, and Steitz, 1985; Zhuang and Weiner, 1986). Inthis study, we have not determined whether sequencevariations in the 5' nucleotides of the U1 snRNAs occur,potentially increasing base-pairing interactions with thehighly variant 5' splice sites present in plant introns(Brown, 1986; Brown, Feix, and Frendeway, 1986; Hanleyand Schuler, 1988). Three of the four dicot U1 snRNAgene sequences available (soybean U1 a, U1 b; broadbeanU1) contain the 10 terminal consensus nucleotides presentin all previously characterized U1 snRNAs (Van Santenand Spritz, 1987; Van Santen, Swain, and Spritz, 1988).The fourth U1 coding sequence (Arabidopsis) has a singlenucleotide insertion within the 5' terminus that increasespotential interaction at intron positions +8 and +9 (A. P.Sturtevant and R.E. Zielinski, in preparation). The limitedset of pea U1 snRNAs that have been sequenced indicatethat minor insertions/deletions at the 3' end of the U1snRNA sequence and between stems III and IV accountfor some of the U1 snRNA size heterogeneities (B.A.Hanley and M.A. Schuler, in preparation).

Pea U2 snRNA consists of a major species, containingthe vast majority of the U2 snRNA, and a minor, smallerspecies. The major U2 snRNA appears to contain a singlevariant of U2 snRNA since, even on our highest resolution15% acrylamide gels, the pea U2 snRNA always migratesas a single prominent species and a minor species (B.A.

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Hanley and M.A. Schuler, in preparation). The six U2 snRNA genes in Arabidopsis, which have been sequenced, contain only minor heterogeneities (Vankan and Filipowicz, 1988). These results collectively suggest that dicot nuclei contain a single or several, highly homologous U2 snRNA variants. The minor form of U2 snRNA (205 nt) present in pea nuclei can be immunoprecipitated with anti-m3G antiserum and 3‘ end-labeled with 3*P-cytidine bisphosphate (data not shown), indicating that this RNA represents an additional form of U2 snRNA expressed at low levels in pea nuclei and not an RNA degradation product. In contrast, five major and two minor wheat U2 snRNA variants (208 to 260 nt) hybridize with the U2 oligonucleotide. Multiple U2 snRNAs with similar size variations occur in each monocot plant that we have examined. The three wheat U2 snRNAs, which have been sequenced, have different primary structures (Skuzeski and Jendrisak, 1985). Thus, each monocot U2 snRNA species probably represents a distinct U2 snRNA variant present in monocot nuclei. Each U2 snRNA variant hybridizes with the U2 oligonucleotide, indicating that all variants contain nucleotides 28 to 42 required for association of the U2 snRNA with the lariat branch point (Black, Chabot, and Steitz, 1985). Thus, heterogeneity in the population lies in other regions of the U2 snRNA, such as those involved in snRNP assembly. Clearly, further sequence analysis is needed to locate the extensive length heterogeneities that exist in the monocot U2 snRNA population. Comparison of the pea and Arabidopsis U2 snRNA indicates that extreme length differences also occur in dicots. But, with the ex- ception of tobacco, which contains two predominant U2 snRNAs, these extreme size differences do not occur within a particular species. Sequence analysis has demonstrated that the majority of the size variations in the broadbean and Arabidopsis U2 snRNAs are due to insertions at the 3‘ end of the molecule that substantially alter the secondary structure of stem-loop IV (Kiss, Antal, and Solymosy, 1987b; Vankan and Filipowicz, 1988). Hypo- thetically, these variations result in the formation of distinct U2 snRNP particles that interact differently with other components of the splicing machinery.

Equivalent sets of U5 snRNAs are resolved in monocot and dicot nuclei. Because this snRNA always fractionates as a diffuse band, we believe that numerous U5 snRNA variants exist in each plant group. The pea U5 snRNAs, which have been characterized, have highly divergent primary sequences (Krol et al., 1983; B.A. Hanley and M.A. Schuler, in preparation). In contrast to the degree of diver- gente in other plant snRNAs, the length of U6 snRNA has been strictly conserved throughout the evolution of monocot and dicot plants. The plant U6 snRNAs (1 O1 to 104 nt) are shorter than the mammalian and Drosophila U6 snRNAs (Reddy, 1988) due to the absence of nucleotides at the 5’ end and numerous small deletions in single- stranded regions of U6 snRNA (Kiss, Antal, and Solymosy, 1987a).

In each plant examined, the U4 snRNA was surprisingly similar in size to the U1 snRNA component. We have resolved the group of 158 to 170-nt snRNAs, originally characterized as the pea U1 snRNA family (Kiss, Toth, and Solymosy, 1985), into multiple U1 and U4 components of nearly equal abundance. At 160 to 176 nucleotides, the most abundant plant U4 snRNAs are significantly larger than their mammalian counterparts (1 45 nt; Reddy, 1988) and more similar in size to the S. cerevisiae U4 snRNA (159 nt; Siliciano et al., 1987). In wheat, we have identified three additional, less abundant U4 snRNAs at higher molecular weights (182 to 192 nt). The U4 snRNA pattern parallels the U2 snRNA profile in that dicots contain a small number of U4 snRNAs that are highly conserved in length. Only on the highest resolution gels can the dicot U4 snRNAs be fractionated into four variants of nearly identical size (B.A. Hanley and M.A. Schuler, in preparation) that represent highly conserved sequence variants (Kiss et al., 1988). In contrast, monocot nuclei contain numerous U4 snRNAs of variable length. The size difference between the most prominent dicot and vertebrate U4 snRNAs results from single nucleotide insertions in the 3’ half of the molecule and additional nucleotides at the 3‘ terminus (Kiss et al., 1988; B.A. Hanley and M.A. Schuler, in preparation). Sequence analysis is needed to determine whether the monocot U4 snRNAs contain similar additions within their sequences.

Clearly, the monocot and dicot snRNA populations are more complex than previously suspected, especially when compared with mammalian and yeast snRNAs. Compari- son of the snRNA populations has also demonstrated that the relative abundance and multiplicity of the U1, U2, and U4 snRNAs vary significantly among plant nuclei. In contrast, U6 snRNA is expressed at consistent levels in all plants. The proportions of U1, U2, and U4 snRNAs present in plant nuclei contrast with the relatively high abundance of U1 and U2 snRNAs in mammalian cells and lower abundance of U4, U5, and U6 snRNAs (Lerner and Steitz,

Trimethylguanosine cap structures occur on all prominent U1, U2, U4, and U5 snRNA variants present in wheat nuclei. As far as we have determined, none of the more abundant species appears to be differentially expressed in leaf development. In the course of our analysis, we discov- ered another nuclear RNA (284 nt) that is substantially larger than the U1 snRNAs previously identified in plants and animals. The higher molecular weight species clearly contains sequences homologous to the 5’ and 3’ termini of U1 snRNAs, and, in contrast to the more prominent snRNA species, this U1 -like RNA is differentially expressed in mature tissue. The U1-like RNA cannot be immunoprecipitated with anti-m3G antiserum, an antiserum that is highly specific for m32,2,7G cap structures and unreactive with m7G, m2G, or m22s2G terminal structures (Luhrmann et al., 1982). Thus, we have concluded that this RNA does not contain a trimethylguanosine cap structure at its 5‘

1979).

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end, but we cannot discount the possibility that this U1- like RNA retains an undermethylated cap structure or represents a U1 snRNA precursor. Further sequence analysis is clearly needed to identify this RNA.

The differences that typify the monocot and dicot snRNA populations indicate that the U1, U2, U4, and U5 snRNAs have evolved independently since the divergence of these two groups of plants 120 million years ago. Some of the snRNA heterogeneities that we have documented, especially in the U2 and U4 snRNA pools, may account for the RNA processing deficiencies encountered when monocot introns are expressed in dicot nuclei (Keith and Chua, 1986). On the basis of our results, we speculate that the small pool of U2 and U4 snRNAs in dicot nuclei may incorrectly interact with the heterogeneous sequences present in monocot introns (Hanley and Schuler, 1988) and block formation of effective RNA splicing complexes.

METHODS

Reagents

-p3’P-ATP (7000 Ci/mmol), cytidine 3’,5’-[5’-3’P]bisphosphate (3000 Ci/mmol), and Gene-Screen transfer paper were obtained from Du Pont-New England Nuclear. All other reagents were analytical grade.

Plant Materials

The plant species used in this comparison are as follows: peas (Pisum sativum Progress No. 9), wheat (Triticum aestivum), Ara- bidopsis thaliana, tobacco (Nicotiana tobacum), alfalfa (Medicago sativa), maize (Zea mays), sorghum (Sorghum vulgare), and barley (Hordeum vulgare). Wheat germ was obtained from Arrowhead Mills (Hereford, TX). For developmental RNA gel blots, 6-cm-tal1 etiolated wheat seedlings were sectioned into thirds. The dista1 section (O cm to 2 cm) contains mature, differentiated cells, the media1 section (2 cm to 4 cm) contains partially differentiated tissue, and the basal section (4 cm to 6 cm) contains dividing and elongating cells.

Preparation of Total and Nuclear RNA

All plant RNA samples, except pea and wheat, were prepared from unfractionated cells (total RNA). The Arabidopsis, tobacco, and barley RNA samples were derived from plants grown under normal greenhouse conditions. All other RNAs were isolated from etiolated seedlings. In all cases, tissue was rinsed in sterile water, blotted dry, and frozen in liquid nitrogen. Tissue (9 g to 80 g) was ground in a mortar and pestle and homogenized for 2 min in a Waring blender with 4 volumes of 50 mM Tris-HCI (pH 9.0), 10 mM EDTA, 0.2 M NaCI, and 4 volumes of phenol: ch1oroform:isoamyl alcohol (50:50:1). The homogenate was shaken for 10 min at 25OC and then centrifuged at 10,OOOg for 1 O min at 4°C. Nucleic acids were precipitated from the aqueous

phase by adding 0.1 volume of 2 M NaCl and 2 volumes of ethanol. The samples were stored at -2OOC overnight and centrifuged at 10,OOOg for 1 O min. The nucleic acids were reprecipitated twice from 0.2 M NaCl with 2 volumes of ethanol and resuspended in 10 mM Tris-HCI (pH 7.4), 1 mM EDTA.

The pea and wheat nuclear RNA samples were prepared from nuclei isolated from etiolated 7-day-old to 9-day-old seedlings. For the nuclear RNA isolation, 40 g of pea epicotyl tissue or 72 g of 6-cm wheat leaves were rinsed thoroughly in cold sterile water containing 0.5% diethylpyrocarbonate. All further manipulations were carried out at 4°C with sterile, chilled glassware. The tissue was homogenized for 30 sec to 60 sec in a Waring blender with 2 volumes of 2 x homogenization buffer (0.5 M sucrose, 50 mM NaCI, 20 mM Mes (pH 6.0), 5 mM MgCL, 0.3 mM spermine, 0.27 mM spermidine trihydrochloride, 20 mM (3-mercaptoethanol, 0.4 mM phenylmethylsulfonyl fluoride) and leupeptin at a final concentration of 1 O pg/mL. The homogenate was filtered through four layers of cheesecloth and the filtrate was centrifuged at 20009 in an HB-4 rotor for 10 min at 4°C. The crude nuclear pellet was washed quickly with 1.3 x homogenization buffer and recentri- fuged at 20009 for 7 min at 4°C. Each washed nuclear pellet was resuspended in 1 mL of 0.3 M NaCI, 20 mM Hepes (pH 7.0), 5 mM MgCI,, 25% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 10 pg/mL leupeptin, frozen in liquid nitrogen, and stored at -70°C. For RNA extraction, nuclei were thawed on wet ice, adjusted to a final concentration of 50 mM Tris-HCI (pH 8.0) and 0.3% SDS, and extracted with phenol: chloroform (1:l) at 25OC. Nucleic acids were recovered from the aqueous phase by ethanol precipitation at -70°C and were reprecipitated twice from 0.2 M NaCl with 2 volumes of ethanol.

Gel Electrophoresis

Denatured RNA samples were electrophoresed on denaturing 10% polyacrylamide (40:2), 8.3 M urea gels containing 1 x TBE buffer [0.1 M Tris, 0.1 M H3B03, 2 mM EDTA (pH 8.3)]. Low- resolution 10% acrylamide gels, 16 cm in length, were electrophoresed at 35 mA until the xylene cyanol tracking dye reached the bottom of the gel. Higher resolution 10% acrylamide gels, 35 cm in length, were electrophoresed at 45 mA until the xylene cyanol reached the bottom of the gel. Prior to loading, RNA samples were diluted with an equal volume of 10 M urea, 0.1% xylene cyanol, 0.1% bromphenol blue, and denatured at 100°C for 1 min.

RNA Gel Blot Analysis

After electrophoresis, native and denaturing gels were stained for 30 min with ethidium bromide in 25 mM sodium phosphate (pH 6.5) transfer buffer and photographed. The RNAs were blotted onto Gene-Screen transfer membranes for 16 hr at 200 mA at 4OC in 25 mM sodium phosphate buffer (pH 6.5). RNA blots were UV-cross-linked for 10 min and prehybridized for 1 hr to 2 hr at 25°C in 6 x SSC [0.9 M NaCI, 0.09 M sodium citrate (pH 7.0)], 5 x Denhardt’s solution (0.1 ?‘O Ficoll, 0.1% BSA, 0.1% polyvinylpyr- rolidone), 0.2% SDS. The filters were hybridized at low stringency (outlined below) for 12 hr to 20 hr with 106 cpm/filter of a 32P- kinased oligonucleotide probe in the prehybridization solution. The U1, U1-3’A, U2, and U6 oligonucleotide probes were hybridized

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642 The Plant Cell

at 22°C; the U4 and U5 oligonucleotide probes were hybridized at 4OC. After hybridization, blots were washed three times for 30 min in 6 x SSC, 0.2% SDS and two times for 30 min in 6 x SSC at 4OC (W, U5) or at 22OC (U1 , U1-3’A, U2, U6), and autoradiographed at -7OOC. In sequential hybridizations, oligonucleotide probes were stripped from the blots by washing two to four times for 20 min to 30 min at 65OC in 50% formamide. 1 x SSC.

Oligonucleotides

The oligodeoxyribonucleotide probes used for these analyses are complementary to particular snRNAs, as outlined in Table 1. An additional oligonucleotide, designated U1-3’A, is complementary to nucleotides 135 to 152 at the 3’ end of plant U1 snRNAs. All oligonucleotides were synthesized at the University of lllinois Biotechnology Center using an Applied Biosystems (Foster City, CA) 380A synthesizer and were gel-purified or HPLC-purified on a C-4 column prior to use. Purified oligonucleotides were kinased with Y-~~P-ATP and T4 polynucleotide kinase and used directly for analysis.

lmmunoprecipitation of Plant snRNAs

Wheat nuclear snRNAs were immunoprecipitated with anti-m3G antisera according to the method of Bringmann et al. (1983) as modified by Krol et al. (1983). Wheat nuclear RNA (0.33 mg) was immunoprecipitated with 150 pg of anti-m3G IgG. The immunoprecipitated RNAs were recovered by proteinase K treatment and phenol extraction, and were redissolved in 25 pL of sterile water. Of this, 1 pL was 3‘ end-labeled with 32P-cytidine bisphosphate and T4 RNA ligase (England, Bruce, and Uhlenbeck, 1980). Twelve microliters of the unlabeled, immunoprecipitated RNA were directly analyzed on RNA gel blots.

ACKNOWLEDGMENTS

We thank especially Dr. JoAnn Wise, Greg Porter, and Patrick Brennwald for providing oligonucleotide probes and Dr. Reinhard Luhrmann for the anti-m,G antiserum. We thank Brian Hanley for extensive technical advice throughout this project. This work was supported by National Science Foundation grant DCB-84-02792 and Public Health Service grant 1-GM39025 R01 from the General Medical Institute.

Received February 21, 1989; revised April 20, 1989.

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molecular analysis of dicot and monocot small nuclear rna

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