a small nuclear ribonucleoprotein associates with the aauaaa polyadenylation signal in vitro

Cell, Vol. 45, 581-591, lWay 23, 1986, Copyright 0 1986 by Cell Press

Small Nuclear R ibonucleoprotein Associate AUAAA Polyadenylation S ignal In V it

Carl Hashimoto” and Joan A. Steitz Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut 06510

~rnrnar~

ntaining the polyadenylation sites for s L3 or E2a mRNA or for SV40 early or late

mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyade-

actions are probed with ribonuclease Tl dies directed against either the Sm pro-

lein determinant or the trimethylguanosine cap structure at the 5’ end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be al-

red, but not always abolished, by pretreatment with icrococcai nuclease. We discuss the involvement of

small nuclear ribonucleoproteins in the cleavage and A) addition reactions that form the 3’ ends of eukaryotic mRNAs.

Small nuclear ribonucieoproteins (snRNPs) containing U RNAs are structurally related RNA-protein complexes found in the cells of higher eukaryotic organisms (Lerner et al., 1980). These snRNPs are recognized by anti-Sm antibodies from patients with autoimmune diseases and are therefore called Sm snRNPs (Lerner and Steitz, 1979). The total number of distinct Sm snRNPs in mammalian cells is currently unknown, but it includes the very abundant (10” copies per cell) Ui and U2 snRNPs, the slightly iess abundant (2 x 105 copies per cell) snRNPs containing U5 RNA and U4/!J6 RNAs, and the low abundance snRNPs containing other U RNAs discovered only recently (Strub et al., 1984; Reddy et al., 1985).

So far the functions of four Sm snRNPs in pre-mRNA processing reactions have been identified and tested ex- perimentally. The sea urchin snRNP containing U7 RNA is required for the formation of the 3’end of sea urchin histone H3 mRNA (Strub et al., 1984). Because U7 RNA shows sequence complementarity to conserved, essential sequences near the 3’ends of histone pre-mRNAs, the suggestion has been made that U7 RNA interacts with these sequences during processing (Strub et al., 1984; Birnstiel et al., 1985). 111 and U2 snRNPs function in pre- mRNA splicing (Krgmer et al., 1984; Padgett et al., 1984;

‘Presen’t address: Department of Molecular Biology, University of California, Berkeley, California 94720.

Black et al., 1985; Krainer and Maniatis, 19 nize specific sequences in the pre-mRNA tant for splicing: the Ul snRNP binds the 5’ splice site, whereas the U2 snRNP associates with the branch point region within the intervening sequence (Mount et al., 1983; Black et al., 1985). Another Sm snRNP (most likely that containing lJ5 RNA) specifically recognizes the 3’ splice site of the pre-mRNA in a splicing extract (Chabot et al., 1985).

Polyadenylation is another pre-mRNA processing event that has been thought to require snRNPs. This two step reaction involves cleavage of the pre-Mona at a specific site and then polymerization of about 200 adenylate residues, poly(A), to the newly generated 3’ end (Darnell, 1982; Moore and Sharp, 1985). Two sequences important for polyadenylation have been identified. The first is a highly conserved hexanucleotide, AAUAAA~ iocated 10 to 30 nucleotides upstream of the A) addition site (Proudfoot and Brownlee, 1976). Del of or point muta- tions in this sequence prevent the appearance of properly polyadenylated mRNA in vivo (Fitzerald and Shenk, 1981; Higgs et al., 1983; Monte11 et Stephenson, 1984; Orkin et al., ). The second is a less conserved sequence downst of the poly(A) addition site (Birnstiel et al., 1985; McLauchlan et ai., 1985). Deletions in this region also inhibit accurate polyadenylation in vivo (e.g., McDevitt et al., 1984; Sadofsky et al., 1985).

Of the U RNAs sequenced so far (Reddy, 1985), U4 RNA shows the best complementarity to the AAUAAA sequence (5/6 match) and some compl~me~tarity to sequences near the poly(A) addition (Benoist et al., 4980; Berget, 1984). Thus, Berget ( ) has proposed that the snRNP containing U4 RNA functions in polyadenylation. Recently, Moore and Sharp (1984, 1985) showed that accurate polyadenylation in vitro is inhibited by anti- Sm antibodies as well as by antisera specific for Ul snRNPs or for the nuclear antigen La, which binds na- scent RNA polymerase III transcripts (Rinke and Steitz, 1982).

We have taken a different approach to defining snRNP involvement in polyadenylation. By probing in vitro polyadenylation reactions with antibodies specific for snRNPs, we have found that a factor with the properties of an Sm snRNP associates with the AAUAAA polyadenylation signal.

esults

fferent RNA Substrates Are Pa! enylated clear Extract

We have made RNA substrates containing the polyadenylation sites for adenovirus L3 or E2a mR early or late mRNA. 32P-labeled RNAs were synthesized in vitro from an SP6 (L3 and E2a) or T7 (SV40 early and iate) RNA polymerase promoter. After incubation for 7 hr in a nuclear extract of HeLa ceils previously described to

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-329 -319 -271 -226

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12345 7 6 9 IQ 11 12 Figure 1. Four Different RNAs Are Substrates for Cleavage and Poly(A) Addition in Nuclear Extract

RNAs synthesized in vitro with SP6 or T7 RNA polymerase were In- cubated in nuclear extract (NE) alone for 1 hr (lanes 2, 5, 8, 11) or in nuclear extract plus the ATP analogue AMP(CH,)PP (AA) for 30 min (lanes 3,6, 9, 12) as described in Experimental Procedures. Extracted RNAs were electrophoresed in a 5% polyacrylamide gel. The starting substrates are shown in lanes 1,4, 7, and 10. Bands marked with dots represent RNAs upstream of the poly(A) addition sites produced by cleavage of substrates. Arrow marks downstream cleavage product of L3 RNA. Downstream cleavage products of other substrates are visible in longer autoradiographic exposures of this gel. Numbers indicate total nucleotides in the substrates. The positions of xylene cyanol (XC) and bromophenol blue (BB) dyes are indicated.

be active in pre-mRNA splicing (Krainer et al., 1984) and To determine whether an Sm snRNP interacts with se- polyadenylation (Moore and Sharp, 1985), each of the four quences important for polyadenylation, we first probed RNA substrates is converted to a form (following phenol reactions with a monoclonal antibody directed against the extraction) that migrates more slowly and diffusely in a 5% Sm protein determinant. Aliquots of a po~yade~y~atio~ polyacrylamide gel (Figure 1, lanes 1 and 2,4 and 5,7 and reaction containing 32P-labeled L3 RNA substrate were 6, 10 and 11). This slower migrating material contains removed at various times and then mixed with the anti- poly(A), as demonstrated by its selective retention on bodies and RNAase Tl at 0% After 30 min of incubation, oligo(dT)-cellulose (data not shown). The efficiency of RNA fragments bound to Sm snRNPs (or antigenic pro- polyadenylation and the stability of RNA precursors and teins) were recovered with protein A-Sepharose and frac-

products in the in vitro system vary among the substrates. (Note that much of the E2a and SV40 early RNAs appear to be degraded by the end of the reaction.)

Moore and Sharp (1985) showed that polyadenylation of an adenovirus L3 RNA substrate similar to ours is quite accurate. By the end of the reaction (3 hr in their case), 75% of the RNA substrate was cleaved and polyadenyi- ated at the site used in vivo, while 20% was inaccurately processed. We have used their hybridizationlribonucle- ase protection assay with an Ml3 cDNA to confirm that our 13 substrate is accurately polyadenylated with similar efficiency (data not shown).

An intermediate in the in vitro polyadenylation reaction is an RNA cleaved at the correct site for poly(A) addition. This intermediate is not normally detected (e.g., Figure 1, lane 2) but, as Moore and Sharp (1985) have shown, it is produced when poly(A) addition is prevented by replac- ing ATP with the analogue a$-methyleneadenosine 5”- triphosphate, AMP(CH,)PP, whose a,b bond is not hydrolyzable. Incubation of the L3 RNA in the nuclear extract with AMP(CHs)PP produces an RNA whose size (about 280 nucleotides; Figure 1, lane 3, band marked with adot) and RNAase Tl fingerprint (data not shown) are those expected of the correctly cleaved intermediate. The other cleavage product, containing sequences downstream of the poly(A) addition site, is also visible in the reaction with AMP(CHa)PP (Figure 1, lane 3, band marked with an arrow). Its size (about 50 nucleotides) and RNAase Tl fingerprint (data not shown) support the con- clusion that it is the corresponding cleavage product (see also Moore and Sharp, 1985).

incubation of the adenovirus E2a, SV40 early, or SV40 late RNA substrate with nuclear extract and AMP(CHa)PP also yields, in each case, an RNA of the size expected for the intermediate with the correct 3’end for poly(A) addition (Figure 1, lanes 6,9, and 12, bands marked with dots). The sets of oligonucleotides produced by RNAase Tl digestion of each of these RNAs (analyzed either by two- dimensional fingerprinting or by electrophoresis in a 200/o polyacrylamide gel; data not shown) are exactly those predicted from their template DNA sequences. Moreover, a longer exposure of the gel in Figure 1 shows a band in each oi the three reactions whose size (50-60 nucleotides) and Tl oligonucleotide pattern (not shown) are those predicted for the downstream cleavage products. We conclude that these RNAs are also substrates for accurate polyadenylation in our system.

NA Fragments Containing the AA~AAA Sequence re Precipitated by Antibodies against the Sm

Determinant on snRNP Proteins

snRNP Interaction with AAUAAA 583

2 3 4

Figure 2. RNA Fragments Containing AAUAAA Are Precipitated by Anti-Sm Antibodies

(A) Aliquots of poiyadenylation reactions containing the L3, E2a, SV45 early, or SV45 late RNAs were taken after 15 min and mixed with RNAase Tl and anti-Sm antibodies. RNA fragments from immunoprecipitates were electrophoresed in 15% polyacrylamide gels. Labeled bands with asterisks aie RNA fragments containing AAUAAA, while numbers refer to their lengths, as determined by secondary analysis and/or mobility (see text). L3 and E2a RNAs were labeled with [II-~“P]CTP; SV45 early and late RNAs were labeled with [u-~~P]GTP (B) Examples of RNAase Tl fingerprinting of RNA fragments containing AAUAAA. L3 RNA was labeled with [u-~‘P]CTP; hence, in the L3 b’ fingerprint, the 15”mer that contains three labeled phosphates is more intense than the G that contains only one labeled phosphate transferred from the adjacent C. E2a f’ produces only an 18-mer spot, although it migrates as a larger oligonucleotide in a 15% polyacrylamide gel (Figure 2A, lane 2); this discrepancy is probably due to the adjacent UG (see Figure 3) which is not labeled by the [cI-~~P]CTP used to make substrate. The 23-mer in the early i* fingerprint is underrepresented in comparison with the lCmer, although both should contain one labeled phosphate; this result is probably due to inefficient transfer of the larger oligonucleotides from the first to the second dimension in the fingerprinting procedure, since, after RNAase Tl digestion, analysis of band i* in a 20% polyacrylamide gel shows equal amounts of the two oligonucteotides. Faint spots are oligonucleotides that form a minor RNA fragment, of similar size to i*, which maps downstream of the poly(A) addition site.

t ionated in a 15% polyacrylamide gel. Shown in Figure 2A, lane 1, are the RNA fragments immunoprecipitated after 10 min of reaction, when the yields of RNA fragments containing the AAUAAA sequence are highest (see Dis- cussion). The RNA bands labeled a*, b*, and c were eluted from the gel and subjected to complete digestion with RNAase Tl and to fingerprint analysis. The most prominent band, b*, produces two spots, G and the oligonucleotide ACACUUUCAAUAAAG (Figure 28, L3 W), compris- ing a 16-nucleotide region that includes the AAUAAA sequence located approximately 20 nucleotides upstream of the poly(A) addition site. Band a* just above this 18mer is a mixture mostly of two fragments (a and a*; see Figure 3), one of which is an extended version of b* that includes an adjacent AG, the other of which is an extended version of fragment c. The fingerprint of band c reveals a single

oligonucleotide, AAUACACG, which maps to the 5’ end of the RNA; yet, when analyzed in a 20% polyacrylamide gel, band c migrates as a lo-mer, which is converted upon Tl digestion to an 8-mer (data not shown). Thus, band c probably represents the fragment GpppGAAUACACG, which contains the cap structure of the S’end of the RNA. Since the synthesis of L3 RNA was primed with the dinucleotide G(Y)ppp(Y)G, and the transcript was labeled with [cx-~~P~CTP, the cap structure is not labeled. Interest- ingly, fragment c is not immunoprecipitated if the L3 substrate is made without the cap structure (data not shown).

To test whether the immunoprecipitation of AAUAAA- containing fragments is general, we similarly analyzed polyadenylation reactions containing the E2a, SV40 early, or SV40 late RNA substrates. For each RNA the pattern of fragments precipitated by anti-Sm antibodies after 10 min

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GPPPGAAUACACGGAAWCGAGCUCGCCCGGGGAUCCGGGGGGAUCACAACCCCACCAUGAACCUUAUURCCGGGGUACCCAACUCCAUGCW~CAGCCCCAGGUACA

c a

E2a

G~~PGAAUACACGGAA~“~~GAGC~UCGCCCGCGAUCCGGCG~GU”GG”GAUGG”G~G~AGCC”G”GGAG”GAAAAC”“CACCGAGCUGCCGCGGA”GG~G”GCC”GAG”“U _.-.- --a h

b “GGGGGG”AAA”AA”CACCCGAGAGUGURCAAA”AAAAA~A”””GC~“““A”“GAAAG”G”~”~~“AG”A~A”“A”“~“A~A”G”“~~AAG”GA~A-AGAAG”G --

d 1” _~ .~~ ~~~_ .

early

I GCA”CACAAA”““CACARR”AAAG~A”“U”“U”~A~”G~A””C”AG”“G”GGU”“G”~~AAA~”~A”~AA”G”A”~““A”~A”G”~“GGA”~

k’ if I

late

“A”““G”GAAA”““G”GAUGC”A”“G~Uu”A”””G”AACCA””A”AAGC”GCAA”AAACAAG”U~~CAA~~~c~~U”~cA”“cA”“““A”G”””~AGG”“~AG~~GGAGG”G”GGGAGG”u”””” ____-~~

--- 7-n* P------------------

q- r-------I

Figure 3. Locations of lmmunoprecipitated RNA Fragments in the Nuceotide Sequence of Each Substrate

Lines below sequences represent bands in gel lanes of Figure 2A. If a gel band actually contains a mixture of fragments, only the fragment with AAUAAA is labeled with an asterisk (e.g., a and a* in L3). The thickness of the line indicates the amount of a fragment as compared with others rn the immunoprecipitate. Yields of fragmentswere generally low. For example, b’ and c each correspond to about 0.4% of L3 substrate in the reaction, while a and a* are each about 0.05%; o*, p, and q from late RNA are each about O.i%-0.15% yield, while m* and n* are about 0.05% each. Poly(A) addition sites are indicated by arrows. The absence of a G at the 5’ends of the early and late SV40 RNA sequences indicates that posttranscriptional capping of these RNAs was not efficient. Adenovirus L3 and E2a sequences are from Akusjlrvi et al. (1981) while SV40 early and late sequences are from Buchman et al. (1980). SP8 sequences are presented by Melton et a!. (1984) and T7 sequences are in Dunn and Studier (1983).

of reaction is shown in Figure 2A (lanes 2, 3, 4). RNAase Tl fingerprint analyses of the labeled bands reveals that, for each RNA, at least one of the prominent bands represents an RNA fragment containing the AAUAAA sequence (Figure 2B, E2a f”, SV40 early i*, SV40 late n*). Be- cause the RNA substrates were not uniformly labeled, the intensities of bands in the gel lanes do not accurately reflect the relative molar amounts of fragments in the immunoprecipitates. However, if the number of labeled phosphates in each fragment is considered, the AAUAAA- containing fragments are well represented relative to other fragments (see Figure 3).

For each of the four substrates, the specificity of the immunoprecipitation of AAUAAA-containing fragments is supported by several lines of evidence. First, most of the

selected fragments represent protected regions, since they are not primary Tl oligonucleotides but instead contain internal G residues; yet, the Tl nuclease concen- tration used in our experiments produces essentiafly complete digestion of the transcripts, as revealed by polyacrylamide gel analysis of the total binding reactions (data not shown, but see Figure 48, lane 4, or Figure 6, lane 5, for a total Tt digest of the L3 substrate). Second, the protected fragments are not selected by normal human se- rum (data not shown) or other autoantibodies (see Figure 4, below); thus, their recovery must be due to binding by anti-Sm antibodies rather than to nonspecific associa- tions with protein A-Sepharose or immunoglobulin. Third, with the SV4O early RNA (Figure 2A, lane 3) we do not ob- serve immunoprecipitation of a fragment containing the

snRNP tnteraction with AAUAAA 585

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Figure 4. RNA Fragment, v * rontaining AAUAAA Are Precipitated by Anti-msG Antibodies but Not by Antk(U2)RNP or Anti-(U1)RNP Anti- bodies

(A) Polyadenylation reactions containing L3 RNA were probed (after 10 min) with RNAase Tl and antibodies, as in Figure 2A. RNA fragments extracted from anti-Sm (lane l), antk(U2)RNP (lane 2), anti-(Ul)RNP (lane 3) and anti-m36 (lane 4) precipitates were electrophoresed in a 15% polyacrylamide gel. a* and b’ are the AAUAAA-containing fragments shown in Figure 3. The increased number of bands in the anti- Sm and anti-m36 precipitates may be due to partial RNAase Tl digestion. More typical patterns are seen in Figure 6A, lanes 1 and 6. (S) Visible RNA fragments migrating near b” in the gel of (A) were analyzed, after RNAase Tl digestion, in another 15% polyacrylamide gel. Fragments from anti-Sm (lane l), anti-(U2)RNP (lane 2) and anti-msG (lane 3) precipitates were compared with an RNAase Tl digest of the entire L3 RNA (lane 4). Numbers correspond to the sizes of the Ti oligonucleotides of L3 RNA; 15’ contains the AAUAAA sequence. Smaller oligonucleotides of L3 are not visible in this autoradiograph.

stream AA~AAA sequence (see Figure 3); the mini- mum size of such a fragment would be 12 nucleotides. Fourth, the immunoprecipitation by anti-Sm of the AAU- AAA-containing fragments reflects a strong association (either direct or indirect), since it withstands challenge by up to a IOOO-fold excess of competitor RNA and by exten- sive dilution (see Experimental Procedures).

Nonetheless, we cannot explain the presence of all fragments in the immunoprecipitates. The two most intense bands produced by the SV40 late RNA (q and r in lane 4 of Figure 2A) are fragments 12 to 16 nucleotides long that map about 30 nucleotides downstream of the poly(A) addition site (data not shown); since this RNA was labeled with [a-32P]GTP, the intensities of these bands partially reflect their high G content (see Figure 3). Fragments h and g of E2a RNA are the same as the 5’ end fragments c and a, respectively, of L3 RNA, since both RNAs share the same 5’SP6 vector sequences (see Experimental Procedures). A 5’ end fragment is not visible in immunoprecipitates of reactions with the SV40 early and late RNAs, perhaps because a cap structure seems to be required for immunoprecipitation of the 5’ end fragment of L3 RNA (see above) and because posttranscriptional capping of the SV40 RNAs may not be as efficient as use of a cap primer (see Experimental Procedures). The positions of all immunoprecipitated fragments within the four substrate RNAs are shown in Figure 3.

RNA Fragments Containing the AAU Are Precipitated by Antibodies agai Trimethylguanosine Cap Structure of U RNAs We also probed the polyadenylation reaction with antibodies specific for the trimethyiguanosine (m3G) cap structure found at the 5’ ends of all U RNAs except U6 (Bringmann et al., 1983). Among RNA fragments from the 13 substrate that are precipitated by anti-m36 antibodies, a band comigrating with b* is seen (Figure 4A, lane 4). When eluted from the gel, digested with Tl, and electrophoresed in a 15% polyacrylamide gel, this fragment yields a 15-nucleotide Tl product containing the AAUAAA sequence (Figure 4B, lane 3), corresponding to the band produced by Tl digestion of b” from the anti-Sm precipitate (Figure 48, lane 1). When the band above b* in the anti-m3G precipitate is analyzed, we find that it corresponds to band a* in the anti-Sm precipitate (data not shown). Thus, the two AAUAAA-containing fragments are precipitated both by anti-msG and by ant&m antibodies, indicating that the factor responsible has a U RNA component as well as an Sm protein determinant.

To determine whether the factor that associates with the AAUAAA region might be the Ul or the U2 snRNP, we used patient sera containing antibodies specific for these two snRNPs to probe polyadenylation reactions containing L3 RNA. The amounts of ante-(~l)R~P and anti- (U2)RNP sera used were found to precipitate Ui or U2 RNA from the nuclear extract as efficiently as the anti-Sm antibodies used in the experiment of Figure 2. Figure 4A, ianes 2 and 3, shows that both anti-(U2)RNP and anti- (Ul)RNP sera yield a different and much fainter pattern of bands than do anti-Sm antibodies as shown in lane 1. One prominent band in both immunoprecipitates appears to comigrate with b* (an AAUAAA-containing fragment), but, upon digestion with RNAase T1, its migratron in a 15% polyacrylamide gel is unchanged (Figure 48, lane 2); it ap- parently corresponds instead to a 1Fnucleotide Tl product that maps near the 5’end of th Figure 48, lane 2). This l%mer is not found in the substrate used

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L3.

1 2 3 4 5 6 7 8 9 10 11 -- Figure 5. The Cleavage Step before Poly(A) Addition Is Inhibited by Micrococcal Nuclease Digestion of Nuclear Extract

(A) Total RNAs in nuclear extract before (lane 1) and after incubation for 30 min at 30% with Ca s+ (lane 2) or Gas+ plus micrococcal nuclease (MN) (lane 3) were electrophoresed in a 10% polyacrylamide gel. Abundant U RNAs contained in Sm snRNPs were identified on the basis of their characteristic migration. (B) Shown is a 5% polyacrylamide gel of polyadenylation reactions with the L3 RNA substrate. Reactions were for 1 hr except when AMP(CHs)PP was added (+AA) to inhibit poly(A) addition (lanes 4-6) in which case reactions were for 30 min. The nuclear extracts in the reactions were either untreated (lanes 1 and 4) preincubated with Gas+ (lanes 2 and 5), or preincubated with Ca*+ and micrococcal nuclease (MN) (lanes 3 and 6). Controls in which extract was preincubated with EGTA and Ca2+ (lane 7) or with EGTA, Ca*+, and micrococcal nuclease (lane 6) are also shown. Lane 9 shows a reaction containing 13% (v/v) normal extract, as compared with reactions containing a mixture of 13% normal extract and 47% extract that was preincubated either with Ca*+ (lane IO) or with Ca2+ and micrococcal nuclease (lane 11). Arrows mark presumptive cleavage products up stream and downstream of the poly(A) addition site. Faint bands near the middle of the lower half of the gel (e.g., lanes 1 and 2) may be tRNAs from the extract that became labeled during incubation with [a-32P]CTP-labeled substrates (see Slack et al., 1985).

by Moore and Sharp (1985) and it is most likely not required for accurate polyadenylation. The faster-migrating prominent band in the anti-(U2)RNP precipitate likewise does not contain the AAUAAA sequence but is one of two 13nucleotide Tl products that map either near the 5’ end of the RNA or about 40 nucleotides upstream of the AAU- AAA sequence. Thus, we conclude that the AAUAAA- containing fragments a* and b* are not detectably precipitated by either anti-(U2)RNP or two different anti-(U1)RNP sera, including one that inhibits polyadenylation in vitro (Moore and Sharp, 1985).

Nuclease Digestion of the Extract inhibits leavage of the Polyadenylation Substrate

The results described above indicate that the factor that associates with the AAUAAA sequence has the properties of an Sm snRNP containing a U RNA, for it is precipitable by both antiBm and anti-m& antibodies. To determine whether the components required for polyadenylation are nuclease sensitive, we treated the nuclear extract with micrococcal nuclease, which requires free Ca2+ for activity (Pelham and Jackson, 1976) prior to the addition of substrate. Incubation for 30 min at 30% with the enzyme extensively degrades the abundant small RNAs in the nuclear extract (Figure 5A, compare lanes 1 and 3) and completely inhibits polyadenylation of L3 RNA (Figure 58, compare lanes 1 and 3). Some polyadenyla- lion activity is lost during preincubation with Ca*+ alone (Figure 5B, lane 2) even though the small RNAs in the extract do not appear degraded (Figure 5A, lane 2). Inhibi- tion is not due simply to the presence of the micrococcal nuclease preparation, since preincubation of the nuclear extract with the enzyme preparation and EGTA, which chelates Ca*+, does not lead to inhibition (Figure 5B, lane 8). Inhibition is also not due to an inhibitor produced by the micrococcal nuclease treatment, since the RNA cleavage activity in a reaction containing only 13% (v/v) untreated extract (our normal reaction contains 60% extract; Figure 5A, lane 1) is not inhibited by the presence of an almost 4-fold excess (47%) of nuclease-treated extract (Figure 58, compare lanes 9 and 11). In this particular experiment, the poly(A) addition activity in the reaction with 13% untreated extract was unusually inefficient, yet one product of the cleavage step, the 50-nucleotide RNA containing sequences downstream of the poly(A) addition site, is still visible in lanes 9 and 11. Moreover, when tested by the more sensitive hybridization/RNAase protection assay (Moore and Sharp, 1985) the reaction in lane 11 shows levels of accurately polyadenylated RNA compara- ble to those in the reaction in lane 9 (data not shown).

To determine if the cleavage step prior to poly(A) addition is sensitive to micrococcal nuclease, we incubated the L3 RNA in a nuclease-treated extract with AMP(CH& PP which prevents poly(A) addition (Moore and Sharp, 1985). Nuclease treatment completely inhibits the cleavage step, as indicated by the absence of the 280 nucleotide RNA upstream, as well as the 50-nucleotide RNA downstream, of the poly(A) addition site (Figure 58, compare lanes 4 and 6). Some cleavage activity is lost during preincubation with Ca2+ alone (compare lanes 4 and 5).

We next checked whether micrococcal nuclease digestion of the nuclear extract affects the precipitation of the AAUAAA-containing fragments by anti-Sm antibodies. Surprisingly, after nuclease digestion, the AAUAAA- containing fragments, a* and b*, from L3 RNA are still precipitated (Figure 6A, compare lanes 1 and 3). One difference, however, is that other bands have increased in intensity or appeared after nuclease digestion. To determine whether these bands represent fragments that contain AAUAAA or other sequences, we digested the RNA in the anti-Sm precipitates to completion with RNAase Tl before electrophoresis in the 15% polyacrylamide gel

snRNP Interaction with AAUAAA 587

123 45 6 7 8

I3 late

1 2 3 4

Figure 6. lmmunoprecipitation of RNA Fragments after Micrococcal Nuclease Digestion of Nuclear Extract (A) Reactions (10 min) containing L3 RNA and untreated extract (lanes 1 and 2) or L3 RNA and micrococcal nuclease-treated extract (lanes 3 and 4) were probed with RNAase Tl and anti-Sm antibodies. RNA fragments from immunoprecipitates were electrophoresed in a 15% polyacrylamide gel before (lanes 1 and 3) or after (lanes 2 and 4) a second RNAase Tf digestion. Ti oligonucleotides from the entire L3 RNA are shown in lane 5; 15’ contains the AAUAAA sequence. Similar analyses were performed with anti-msG antibodies on an untreated extract (lane 6), an extract preincubated with Ca*+ (lane 7), or an extract preincubated wi?h Caa+ plus micrococcal nuclease (lane 8). (6) Reactions (10 min) containing SV40 late RNA and untreated extract (lanes I and 3) or nuclease-treated extract (lanes 2 and 4) were probed with RNAase T? and antiSm (lanes 1 and 2) or anti-maG (lanes 3 and 4) antibodies. RNA fragments from immunoprecipitates were electrophoresed in a 15% polyacrylamide gel. m: n: and o* are fragments containing the AAUAAA sequence.

(Figure 8A, lanes 2 and 4). Although several Tl oligonucleotides of L3 RNA (lane 5) are present in the anti-Sm precipitate from the untreated extract, the oligonucleotide containing the AAUAAA sequence (labeled 15*) is the most prominent (lane 2). After preincubation of the nuclear extract with micrococcal nuclease, the amount of ~~~go~ucleQtide 15* appears unchanged, but the intensi-

ties of the other, especially larger, ol~~o~ucleotides increase (lane 4). Similarly, when anti-msG antibodies are used, the AAUAAA-containing fragments a* and b* are still precipitated after micrococcal nuclease digestion of the extract (Figure 6A, compare lanes 6 or 7 and 8).

lmmunoprecipitation after pretreatment of the nuclear extract with micrococcal nuclease has also been performed with the other RNA substrates. In contrast to the observations with the L3 RNA, the immunoprecipitation of AAUAAA-containing fragments from the SV40 late RNA substrate by either antiBm or anti-m& antibodies is significantly altered after micrococcal nuclease predigestion of the extract (Figure 6B). Most notably, bands m* and fl* are significantly reduced in intensity; compare lane 2 with lane 1 and lane 4 with lane 3. A band that could be o* is still visible in both immunoprecipitates after nuclease digestion (lanes 2 and 4), but its identity was not determined. Interestingly, the intense bands q and r, which are not precipitated by anti-msG antibodies (Figure 6B, lane 3), remain immunoprecipitable by anti8m antibodies after nuclease digestion (Figure 6B, compare lanes 1 and 2). The results for the SV40 early RNA are yet different from those just described for the L3 and SV40 late RNAs. After nuclease digestion, anti-Sm antibodies still precipitate the AAUAAA sequence (bands i* and k* in Figure 2A), but anti-msG antibodies do not (data not shown).

Our results suggest that a factor with the properties of an Sm snl?NP associates with the AAUAAA signal that is upstream of the polyadenylation site in mRNA precursors. When in vitro polyadenylation reactions are probed with RNAase Tl and antibodies specific for Sm snRNPs, protected RNA fragments containing the AA~AAA sequence are immunoprecipitated from four different substrates (Figures 2 and 3). Binding of these fragments is obtained with both anti-Sm and anti-msG antibodies but not with antibodies specific for Ul or U2 snRNPs (Figures 4 and 6). Although the binding of fragments containing the AAU- AAA signal is not exclusive, specificity is demonstrated by the finding with the SV40 early transcript that oniy the AAUAAA sequence immediately preceding the polyadenylation site is selected (Figures 2 and 3). ments extend to include the cleavage site in only one transcript, the SV40 late RNA (Figures 2 and 3).

Several observations suggest that a specific association with the AAUAAA sequence correlates with the func- tioning of the Sm snRNP at some step of the cleavage/poly(A) addition reaction. First, in earlier studies (unpublished data) we tried to detect interactions between the RNA substrates used here and snRNPs purified bio- chemically (Hinterberger et al., 1983) or immunoprecipitated from cell extracts. Our experiments were modeled on those of Mount et al. (1983), who found that purified Ul snRNPs can bind the 5’splice site of a naked RNA. How- ever, we were unable to detect any interaction with a specific sequence that is found in all four RNA substrates. Only by probing a nuclear extract capable of accurately polyadenylating these substrates have we been able to

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detect a consistent interaction with the AAUAAA sequence. The same strategy of probing an in vitro splicing reaction with antibodies has been used to detect an interaction between the U2 snRNP and the intron branch point region of a pre-mRNA substrate (Black et al., 1985). In addition, the AAUAAA-containing fragments usually are barely detectable in anti8m precipitates generated at the start of the polyadenylation reaction, but they are clearly visible by the next time point assayed (10 min) when polyadenylation is proceeding; thereafter, they decrease in intensity as more of the substrate is processed or degraded (data not shown). Thus, it seems that the association of the snRNP with the AAUAAA sequence is coupled to the active process of polyadenylation.

The association of the snRNP with the AAUAAA sequence during polyadenylation is different from the binding of the Ul snRNP to the E/splice site, because the latter interaction is detected equally at the start of and during the splicing reaction (Black et al., 1985). However, as with the Ul snRNP (Mount et al., 1983), interaction with the pre-mRNAprocessing signal (here, AAUAAA) occurs both in the absence of exogenous ATP (although the extract may contain ATP not removed during dialysis) and when the RNA substrate lacks a S’cap structure (assayed at 10 min of reaction with the L3 RNA; data not shown).

Our studies do not reveal at which step(s) of the reaction-cleavage, poly(A) addition, or both-the snRNP associated with the AAUAAA sequence functions. Other studies suggest that the primary role of the AAUAAA sequence is to specify the site of cleavage, but they have not ruled out some requirement for the hexanucleotide in poly(A) addition (Higgs et al., 1983; Monte11 et al., 1983; Wickens and Stephenson, 1984; Orkin et al., 1985). Moreover, it is known that the artificial joining of a poly(A) addition site containing the AAUAAA sequence to the 3 end of a nonpolyadenylated RNA allows in vitro poly(A) addition to the new 3’ end of the RNA without any detectable cleavage (Manley et al., 1985). We have observed snRNP association with the AAUAAA sequence even when poly(A) addition is blocked with AMP(CH*)PP (data not shown), which indicates that the interaction does not require poly(A) addition. If the immunoprecipitation assay is performed without the addition of RNAase Tl, a small fraction (about 3%) of substrate is precipitated by anti8m antibodies at the start of the reaction. Later, when cleavage or poly(A) addition is detected, the substrate, upstream cleavage product, and polyadenylated RNA are all precipitated with similar low efficiency. Thus, it seems that the snRNP binds either directly or indirectly to the substrate, the intermediate, and the product, all of which contain the AAUAAA sequence.

Sequences downstream of the poly(A) addition site have also been identified as important for the cleavage reaction. However, this region appears not to be essential for the snRNP interaction characterized here, since the same AAUAAA-containing fragments are immunoprecipitated from an L3 RNA that has only 10 nucleotides (compared with the normal substrate, which has 49 nucleotides) downstream of the poly(A) addition site. This result is, perhaps, not surprising, since a small fraction of the L3

RNAcontaining only IO nucleotides beyond the cleavage site is accurately processed in vitro (see Figure 9 in Moore and Sharp, 1985). On the other hand, certain of our observations hint that some other factor(s) may interact with downstream sequences. Fragments containing sequences downstream of AAUAAA are precipitated by anti- Sm antibodies from both the SV40 early and late RNAs (Figure 3). In the case of the SV40 late substrate, some fragments contain both downstream sequences and the AAUAAA signal (which lies unusually close to the cleavage site). Hence, it seems likely that the snRNP interacts with another factor that binds downstream sequences. ln- terestingly, deletion of downstream sequences within fragments q and r from the SV40 late substrate (Figure 3) significantly reduces accurate polyadenylation of SV40 late RNA in vivo (Sadofsky et al., 1985). Also, the SV40 early substrate contains two AAUAAA sequences about 25 nucleotides apart, but only the one nearest the poly(A) addition site is immunoprecipitated (Figure 3) which suggests that the AAUAAA sequence itself is not sufficient for snRNP association.

Our results indicate that a nuclease-sensitive component is required for cleavage of a polyadenylation substrate (Figure 5). Whether this component is the snRNP that associates with the AAUAAA sequence is uncertain, because the association with the hexanucleotide is not blocked in every case by pretreatment of the nuclear extract with micrococcal nuclease. In experiments with the 13 RNA substrate, nuclease digestion of the extract seems only to increase the immunoprecipitation of fragments containing sequences other than AAUAAA (Figure 6A). This could be due to damaged snRNP particles or to released snRNP proteins, both of which probably have general RNA binding activity (Mount et al., 1983). In contrast, the same experiment performed with the SV40 late RNA substrate shows altered association with the AAU- BAA sequence. The larger AAUAAA-containing fragments m* and n* from this substrate encompass several G residues that are distant from the hexanucleotide (Figure 3) and yet are protected from RNAase Tl digestion (Figures 2A and 68). After nuclease digestion of the extract, these G residues are no longer protected (Figure 6B), which suggests either that the snRNP associates with the AAUAAAsequence differently or that it no longer interacts with factors that bind neighboring sequences during the polyadenylation reaction. Thus, it is possible that nuclease digestion abolishes the ability of the snRNP to promote RNA cleavage (either directly or indirectly) but not its ability to associate specifically with the substrate. An analogous situation has been observed with the §m snRNP that binds the 3’splice site during pre-mRNA splicing; specific binding is highly resistant to micrococcal nuclease (Chabot et al., 1985) whereas splicing itself is completely blocked (Krainer and Maniatis, 1985).

It should be possible to determine whether the association of the snRNP with the AAUAAA sequence is direct or indirect when the identity of the snRNP becomes known. Cur inability to precipitate the AAUAAA sequence with anti- or anti-(U2) antibodies does not necessarily mean that the Ul or U2 particle is not involved in polyade-

snRNP interaction with AAUAAA 589

nylation. e used two different patient anti-(Ul)RNP sera, one of which inhibits polyadenylation in vitro (Moore and Sharp, 1984, 1985). If the Ul snRNP does participate in polyadenylation, then the inhibition of polyadenylation may reflect the binding of antibody to a functionally important site on the Ul snRNP that becomes inaccessible when components have assembled on a polyadenylation substrate.

In further attempts to identify which snRNP functions in polyadenylation, we assayed the effect of digesting specific U RNAs in the nuclear extract with complementary oligodeoxynucleotides and RNAase H (Kramer et al., 1984; Black et al., 1985; Krainer and Maniatis, 1985). Cligodeoxynucleotides that selectively target Ul, U2, U4, or US RNA for digestion were used (Black et al., 1985; unpublished data). We find that neither RNA cleavage nor

y(A) addition is inhibited when any one of these U As is destroyed or when both U4 and U6 RNAs are

digested. However, since a small fraction (5%-10%) of each U RNA remains undigested after incubation of the nuclear extract with oligodeoxynucleotide and RNAase H, we cannot conclude from these experiments that the abundant snRNPs containing Ul RNA, U2 RNA, or U4 and U6 RNAs are not required for accurate polyadenyl- atiOn.

It is possible that the snRNP that associates with the AAUAAA sequence is a nonabundant snRNP (Reddy et al., 1985) like the snRNP containing sea urchin U7 RNA, which functions in the formation of histone H3 mRNA 3’ ends (Strub et al., 1984). Clearly, the focus of future work will be the identification and characterization of this snRNP It will also be of interest to assay the association of this snRNP with point mutants of the AAUAAA sequence (e.g., Wickens and Stephenson, 1984) that cannot direct normal levels of accurate polyadenylation.

Experimental procedures

Plasmids and RNA Synthesis An adenovirus DNA fragment containing the L3 and E2a polyadenylation sites (on opposite strands) was removed from mpAH12 (from C. Moore, MIT; Moore and Sharp, 1984) by digestion with Smal (site in Ml3 DNA) and Mlul (nucleotides 1036-1041 in adenovirus DNA; Akus- j&vi et al., 1981). After the attachment of BamHl linkers, this fragment was inserted in either orientation into the BamHl site of pSP65 (Melton et al., 1984) to produce spL3 or spE2a. SV40 DNA (from D. Solnick, Yale University) containing the early and late polyadenylation sites (on opposite strands) between the unique BamHl and Bell sites (Buchman et al., 1980) was inserted in either orientation into the BamHl site of pAR864 (from F. W. Studier, Brookhaven; pAR864 contains T7 DNA, position 57.23-57.40, according to Dunn and Studier, 1983, inserted into the BamHl site of pBR322, as described by Studier and Rosen- berg, 1981) to produce pA9 (early poly(A) site) or pAl5 (late poly(A) site). For making transcription templates, spL3 was digested with Ahalll or Dral, spE2a with Haell, pA9 with BamHI, and pA15 with Ahalll or Dral. To produce a template for synthesis of an L3 RNA containing only IO nucleotides downstream of the poly(A) addition site, spL3 was incubated with Hpatl methylase (to methylate an upstream Aval site) and then digested with Aval.

Transcription with SP6 RNA polymerase (10 units, Boehringer- Mannheim) was done for 1 hr at 40DC in 25 ~1 containing 2 ftg plasmid DNA, 0.1 mM each of four nucleoside triphosphates, 20-100 &i :@P]CTP (410 Cilmmol, Amersham), and the buffer described by Melton et al. (1984). The dinucleotide G(5’)ppp(5’)G (0.5 mM, Pharma- cia PL) was present to serve as a primer for the synthesis of capped

transcripts (Konarska et al., 1984). For synthesis of transcripts of higher specific activity, the unlabeled CTP was omitted from the reaction. Transcription with T7 RNA polymerase (62.5 units, US Biochemical Corp.) was done for 30 min at 37“C in 25 &I containing 2 kg plasmid DNA, 0.4 mM each of four nucleoside triphosphates, 50 j&i [a-3zP]CTP or [03*P]GTP, 40 mM Tris-HCI, pH 8, 20 mM MgCis, and 5 mM DTT All RNAs were purified by electrophoresis in a 5% acrylamide/0.25% bisacrylamide gel containing 8.3 M urea and 90 mM Tris, 90 mM boric acid, 2.5 mM EDTA (TBE); gel slices were eluted with 0.5 M sodium acetate, 1 mM EDTA, and 0.1% SDS. RNAs were stored in 30% ethanol. RNAs synthesized with T7 RNA polymerase were subjected after purification to a‘%apping”reaction with vaccinia virus guanylyltransferase (8 units, BRL) for 1 hr at 37OC in 50 pl containing 50 mM Tris-HCI, pH 7.9, 1.25 mM MgCIz, 6 mM KCI, 2.5 mM DTT, 50 mM GTP, 50 mM S-adenosylmethionine, and 30 units RNasin (Promega-Biotec) (Mon- roy et al., 1980).

Nuclear Extract and Polyadenylation Reaction Extracts of nuclei from HeLa cells were prepared as described by Dig- nam et al. (1983); their dialysis buffer D (20 mM Hepes-KOH, pH 7.9, 20% [v/v] glycerol, 0.1 M KCI, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) was used.

Polyadenylation reactions were incubated for various times at 30°C in 25 pl containing 2.6% (w/v) polyvinyl alcohol, 1 mM ATP, 20 mM creatine phosphate, 12%-60% (v/v) nuclear extract, and 32P-labeled RNA (2 ng or more). When the amount of nuclear extract was below 60%, a compensating amount of buffer D was added. If the ATP analogue AMP(CH2)PP (Pharmacia PL) was added at ? mM, both ATP and creatine phosphate were omitted. Reactions were usually stopped by the addition of 210 pl of buffer containing 0.2 mglml proteinase K (Beckman), 0.5% SDS, 0.2 M NaCI, 10 mM Hepes-NaOH, pH 7.6, and 1 mM EDTA; reactions were incubated 30 min further at 30aC. RNAs were extracted with phenol:chloroform:isoamylaicohol (50:50:1) containing 0.1% hydroxyquinoline (PCA), precipitated with ethanol, and fractionated by electrophoresis in a gel containing 5% acrylamide/ 0.25% bisacrylamide, 8.3 M urea, and TBE buffer. Gels were usually exposed to XAR or XRP film (Kodak) at room temperature.

RNAase Tl Digestion, Immunoprecipitation, and Fingerprint Analysis Fifteen or twenty-five microiiter aliquots of a polyadenylation reaction (containing 3-7 x lo5 cpm RNA substrate) were removed at various times and mixed with IO II antibodies (about 10 to 20 mglml IgG) and 5 pl RNAase T! (30 unitslpl, Calbiochem) at OOC. After 30 min, 50 pl of protein A-Sepharose (4 mg, Pharmacia) in NET-2 buffer (150 mM NaCI, 50 mM Tris-HCI, pH 7.5,0.05% NP40 (Calbiochem]) was added. Usually, 4-10 pi of yeast RNA (IO mglml, Worthington) or E. coil l&S rRNA (10 mglml, from P Moore, Yale University) was added simulta- neously. After an additional 20 min at 0°C with frequent but gentle mix- ing, the protein A-Sepharose beads were washed three to four times, each time with 1 ml NET-2 buffer. RNA fragments were extracted with PCA, precipitated with ethanol in the presence of 14 pglml yeast RNA (5 to 20 gg total), and fractionated by electrophoresis in a gel of 15% acrylamide/0.75% bisacrylamide, 7 M urea, and TEE buffer. For preparation of RNA fragments for fingerprinting, samples from 6-12 reactions were combined just before gel electrophoresis.

RNAs were eluted from gels in 0.5 ml of 0.5 M sodium acetate, 1 mM EDTA, 0.1% SDS, and 40 vg/ml yeast RNA. After precipitation with ethanol, RNAs were digested with RNAase Tl and subjected to fingerprint analysis as described by Barrel1 (1971); for the second dimension, thin layer chromatography on Cel PEI 300 plates (Brinkmann) with homo- mix C was used. Tl oligonucleotides were eluted from PEI plates and analyzed by digestion with RNAase A (Calbiochem) and electrophoresis on DEAE paper at pH 3.5. RNAs were also anaiyzed, after RNAase Tl digestion, by electrophoresis in gels of 15% acryiamidel0.75% bisacrylamide or 20% acrylamide/l% bisacrylamide, containing 7 M urea and TBE buffer.

Antibodies from patient sera and ascites fluid were prepared by am- monium sulfate precipitation, as described by Mimori et al. (1984). We used the following antibodies: Y12 (mouse monocionar anti-Sm; Lerner et al., 1981); Ya (anti-(U2)RNP; Mimori et a!., 1984); Ag and Do (anti- (Ul)RNP; Padgett et al., 1984; Pettersson et al., 1984); and anti-m3G (Chabot et al., 1985).

Cell 590

Micrococcal Nuciease Digestion Fifteen microliters of nuclear extract was mixed with 1 pi 5 mM CaCIz and 1 ~1 of 20 unit&d micrococcal nuclease (Cooper Biomedical) for 30 min at 30%. To stop the reaction, 1 ~1 5 mM EGTA was added. For assaying polyadenylation, polyvinyl alcohol, ATP, creatine phosphate, and RNA substrate were added to the concentrations described above for 25 ~1 reactions. Small RNAs isolated from extracts, as described above, were electrophoresed in a gel of 10% acrylamide/0.38% bisacrylamide, 7 M urea, and TBE buffer; stained for 10 min with 0.5 pglml ethidium bromide; and visualized over short wavelength UV light.

We are grateful to Claire Moore and Phillip Sharp and to Doug Black and Benoit Chabot for sharing materials and information that made many of our experiments possible. For helpful comments on the manuscript, we are grateful to Manny Ares, Doug Black, Benoit Chabot, Nouria Hernandez, Kimberly Mowry, and Sandy Wolin. We thank Donna Villano for help in preparing the manuscript. This work was supported by grant GM 26154 from the National Institutes of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 12, 1985; revised March 11, 1986.

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Mote Added in Proof

L. C. Ryner and J. L. Manley (manuscript submitted) have recently discovered that the inhibition of cleavage and polyadenylation caused by micrococcal nuclease pretreatment of the extract (see Figure 5) may be due to a general requirement for mass of RNA, since processing can be restored by addition of RNA molecules from various sources.

a small nuclear ribonucleoprotein associates with the aauaaa polyadenylation signal in vitro

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