(Received for publication, July 22, 1996, and in revised form, October 4, 1996)
From the Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
The U6 small nuclear RNA is
post-transcriptionally processed by the addition and removal of
nontemplate uridylates (Us) at its 3 end prior to incorporation into
the U4·U6 small nuclear ribonucleoprotein complex. An enzyme
responsible for removing Us from the U6 3
end has not been previously
identified. Here we biochemically isolate and characterize an
exonuclease activity from HeLa cells that removes template and
nontemplate 3
-nucleotides specifically from U6 RNA. We also report the
isolation of an inhibitor of this U6 nuclease. U6 nuclease rapidly
removes the four terminal 3
Us found in the human U6 coding sequence
in a magnesium-dependent manner. Mutagenesis studies on the
U6 RNA define regions essential for processing. U6 nuclease recognizes
specific sequences on both strands near the base of the major
intramolecular stem loop of the U6 RNA. The preprocessed 3
Us form
part of the base of the stem loop, but neither specific sequences nor
secondary structure at the four terminal nucleotides are required to
achieve processing.
RNA polymerase (pol)1 III produces
numerous small RNAs that have a variety of functions (1, 2). pol III
transcripts include tRNAs, adenovirus VA1 RNA, U6 snRNA, 5 S rRNA, and other RNAs such as B1-Alu, which might be
involved in retrotransposition of short interspersed elements. One
common feature of all these RNAs is a tract of uridylates (Us) at their
3 ends that signals pol III to terminate transcription. Apart from pol
III termination, these oligo(U) tracts have no known function, although
the B1-Alu tail might hybridize with an internal A tract to
provide a primer for reverse transcriptase (3, 4, 5).
In a number of documented cases, large portions of the 3 end of
certain pol III transcripts are removed during the course of
transcription by the pol III transcription complex (6, 7, 8). Binding of
the nuclear autoantigen La protein to the oligo(U) tract of the nascent
transcript appears to stabilize it against processing. In other reports
of shortened transcripts, the last few nucleotides are absent, which
might be due to pol III pausing (9). In this case, the La protein
promotes proper termination and transcript release (9).
The U6 snRNA transcript undergoes major 3 end processing in
vivo before it is incorporated into the U4·U6 snRNP splicing complex, and the 3
region is essential for splicing function (10). At
the completion of U6 synthesis, the La protein associates with the 3
uridylate tract of U6 (UUUU-OH) as long as it contains a
3
-OH (11). What role the La protein serves in binding to the released
transcript is not known, although it may stabilize U6 against
degradation. The 3
end of La-associated U6 becomes heterogeneous
through nontemplate 3
addition of Us (11, 12, 13). What happens next is
unclear; however, the 3
end is eventually processed back to five Us in
which the terminal U contains a 2
,3
-cyclic phosphate (UUUU(U)>P)
(14). The activity responsible for the generation of the 2
,3
-cyclic
phosphate has not been biochemically identified. Formation of the
2
,3
-cyclic phosphate is thought to release the La protein, thereby
allowing the processed U6 RNA to become associated with the U4·U6
snRNP. Most of the processed U6 RNA, however, is not associated with
either snRNP complexes or the La protein (11).
While U6 transcripts have been observed to be shortened by a nuclease
in vitro (15), there has been no reported biochemical characterization of a nuclease that specifically targets U6 RNA for
removal of its 3 Us. Here we report the isolation and initial characterization of a 3
-exonuclease that is specific for the U6 snRNA.
We address whether the shortened U6 transcript is generated transcriptionally or post-transcriptionally. We assess how many nucleotides are removed and whether the removal is exo- or
endonucleolytic. We identify from which end of the RNA the nucleotides
are removed. Evidence for an inhibitor of the U6 nuclease is presented,
and the kinetics of processing are examined. Finally, through
mutagenesis of the U6 RNA we examine the specificity determinants of
the U6 nuclease, both for U6 recognition and nucleotide removal.
H buffer contained 20 mM HEPES, pH 8, 5% glycerol, 2 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, and KCl. For all buffers in the H series, the number after the H denotes the molar KCl concentration. TSB+ contained 20 mM Tris acetate (50% cation, pH 8), 5% glycerol, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 200 mM potassium glutamate, 0.1% Nonidet P-40, and 0.1 mM phenylmethylsulfonyl fluoride. The plasmid templates pU6 (human U6 snRNA promoter), pBRVA1, and G6TI have been previously described (16, 17, 18). Oligonucleotides were synthesized using the Oligo 1000 DNA synthesizer (Beckman Instruments). T7 RNA polymerase and Taq DNA polymerase were purchased from New England Biolabs and Promega, respectively. HeLa nuclear extracts were prepared according to Dignam et al. (19), with slight modification (20). The human TATA binding protein was prepared as described (21).
U6 Nuclease PurificationHeLa nuclear extracts (1.3 g, 50 ml) were dialyzed to a conductivity equivalent to H.15 buffer and
chromatographed on phosphocellulose (75-ml column), which was
equilibrated with H.15 (18). Proteins were step eluted with buffers H.3
and H.5, and designated P.3 (130 mg, 90 ml) and P.5 (70 mg, 90 ml),
respectively. Each fraction was dialyzed against TSB+ buffer. Fractions
to be purified further were dialyzed against H.1 buffer. The P.5
fraction (35 mg, 45 ml) was loaded onto a 1-ml Mono Q fast protein
liquid chromatography column equilibrated with H.1 buffer at a flow
rate of 0.5 ml/min. After washing the resin with H.1 buffer, the column
was developed with a 20-ml linear gradient from 100 to 600 mM KCl in H buffer. Fractions (1 ml) containing U6 nuclease
activity eluted between 360 and 390 mM KCl (3.5 mg, 8 ml).
At this stage U6 nuclease was purified by nearly 200-fold. Active
fractions were pooled, dialyzed against TSB+, and stored at 80 °C.
Substantial purification of U6 nuclease was achieved by Mono S
chromatography, eluting between 300 and 500 mM KCl.
However, processing assays performed with the Mono S fractions
displayed an unacceptably high level of nonspecific RNase activity and
thus were not used for characterization.
The P.3 fraction was
generated as described for the U6 nuclease purification and was
precipitated with 0.42 g/ml ammonium sulfate. The precipitate was
collected by centrifugation, solubilized with H.1 buffer, and dialyzed
to a conductivity equivalent to H.1. A portion of the concentrated P.3
fraction (~20 mg, 1 ml) was loaded onto a 1-ml Mono Q column, washed
with H.1 buffer, and eluted with a 130-ml linear gradient from 100 to
600 mM KCl in H buffer. The U6 nuclease inhibitor eluted
over six 1-ml fractions between 280 and 320 mM KCl. Active
fractions were dialyzed against TSB+ and stored at 80 °C.
The polymerase chain reaction (PCR) was used to fuse the T7 promoter to the U6 coding region. Primer combinations and sequences are shown in Table I. PCR reactions contained ~0.5 µM primer, 0.4 mM dNTP, 0.2 ng/µl pU6 plasmid, 2.5 units of Taq polymerase, 5% dimethyl sulfoxide, and 10% glycerol. PCR cycles consisted of 1 min at 94 °C, 2 min at 42 °C, and 2 min at 72 °C. The resulting PCR products of the appropriate size were gel purified by standard techniques and stored in 10 mM Tris-Cl and 0.1 mM EDTA. For the deletion mutants, the range of nucleotides present in the mutant relative to the wild type is defined within the parentheses, e.g. U6(1-76) contains wild type nucleotides 1-76. Point mutants are designated with the wild type sequence first, followed by the nucleotide position and then the nucleotide substitution. For mutants containing a tract of substitutions only the first nucleotide position is indicated; e.g. U4103A4 has four As substituted for four Us starting at position 103.
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Except where noted, all pol II and III
transcription reactions were performed under identical conditions: 5 mM HEPES, pH 8, 1 mM Tris-Cl (pH 8), 1%
polyvinyl alcohol, 60 mM KCl, 60 mM potassium glutamate, 0.5 mM spermidine (G6TI reactions
used 4 mM), 3 mM MgCl2, 10 µg/ml
poly(dG-dC), 1 µg/ml promoter DNA, 5% glycerol, 0.05 mM
EDTA, 0.5 mM dithiothreitol, 0.5 mM GTP, 0.5 mM CTP, 0.5 mM UTP, 10 µM ATP,
and 5 µCi of [-32P]ATP. The reactions also contained
protein fractions as indicated in each figure. When present, and except
where indicated, the following amounts of proteins were typically used:
25-75 µg of nuclear extract, ~5 µg of P.3, ~2 µg of P.5, and
3 ng of TATA binding protein (for U6 reactions only). All reactions
performed with the U6 template contained 2 µg/ml
-amanitin.
Reactions were performed in a total volume of 20 µl and were
preincubated for 20 min at 30 °C before the addition of NTPs.
Reactions were allowed to proceed for 30 min and then terminated with
80 µl of stop mix (3.125 M ammonium acetate and 125 µg/ml tRNA). The RNA was extracted with a phenol/chloroform mixture,
precipitated with ethanol, resuspended in 90% formamide, and
electrophoresed on 7 M urea, 6% polyacrylamide gels. Dried
gels were subjected to autoradiography with an intensifying screen.
Reconstituted reactions were routinely tested for sensitivity to high
concentrations of
-amanitin to verify that they were pol III
transcripts.
T7-synthesized RNAs were generated at 30 °C for 45 min in reactions containing 4 mM Tris-Cl (50% cation, pH
8), 6 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol, 0.5 mM GTP, 0.5 mM CTP, 0.5 mM UTP, 5 µM ATP, 10 µCi of [-32P]ATP, 0.4 ng/µl T7-U6 DNA template,
and 25 units of T7 RNA polymerase in 25 µl. The RNA was
phenol/chloroform-extracted and ethanol-precipitated. Precipitates were
solubilized in diethyl pyrocarbonate-treated double distilled
H2O and stored at
20 °C.
Uniformly 32P-labeled RNA was incubated with the U6 nuclease fraction as indicated in each figure. Reactions were performed at 37 °C and also contained 6 mM Tris acetate (pH 8), 65 mM KCl or potassium glutamate, 6 mM MgCl2, 6% glycerol, 0.03 mM EDTA, 0.3 mM dithiothreitol, and 0.03 mM phenylmethylsulfonyl fluoride in a volume of 10 µl. Reactions were terminated with an equal volume of 0.8% sodium dodecyl sulfate, 20 mM EDTA, and 50% formamide. Reaction mixtures were then lyophilized to approximately 2-3 µl and electrophoresed on 7 M urea, 8% polyacrylamide gels. Gels were vacuum dried and subjected to autoradiography using a PhosphorImager (Molecular Dynamics).
Transcription of
the 106-nucleotide human U6 snRNA gene can be reconstituted in HeLa
nuclear extracts (Fig. 1A, lane
1). The majority of the transcript migrated several nucleotides
shorter than expected (denoted by the large arrow). A minor
amount migrated at the expected size (small arrow). The
production of both transcripts required the U6 promoter and was
inhibited by high but not low concentrations of -amanitin (data not
shown), which indicated that the transcripts were U6- and pol
III-derived. The two transcripts could be due to alternative pol III
start or stop sites or could be due to post-transcriptional processing
of the U6 RNA.
The presence of alternative start sites is unlikely to account for the two transcripts since prior studies have demonstrated only a single start site for the U6 gene (22, 23). pol III will pause near the end of the gene when the La protein is absent (9). The La protein allows for efficient and accurate termination of transcription. Inasmuch as the La protein is present in nuclear extracts (24, 25, 26) and we fail to observe such pausing at other pol III genes such as VA1 (Fig. 1A, lane 2, and data not shown), pausing is unlikely to account for the shortened transcript, although this possibility cannot be ruled out. The shorter U6 transcript is unlikely to be caused by a nonspecific RNase, since other pol III- and pol II-derived transcripts were not shortened (lanes 2 and 3).
In an effort to identify the source of the shortened U6 transcript, nuclear extracts were passed over phosphocellulose and step eluted with 0.3 and 0.5 M KCl (P.3 and P.5 fractions, respectively). The P.5 fraction reconstituted U6 transcription (although, in general, only weakly) but yielded only the shorter transcript (Fig. 1B, lane 3). Comparison to size standards on a high resolution sequencing gel revealed a distribution of transcripts having a median size of 101 nucleotides. When increasing amounts of the P.3 fraction were included in the U6 transcription reaction, longer transcripts centered around 106 nucleotides were observed (lanes 1 and 2). This suggested that a factor present in the P.3 fraction inhibited the production of the shorter transcripts. This inhibitor was purified further by Mono Q chromatography. As shown in Fig. 1C, an activity eluting across fractions 15-19 (280-320 mM KCl) inhibited the production of the shorter U6 transcripts. From these data we conclude that a distinct biochemical entity inhibits the production of shortened U6 transcripts. At this point we do not know if this inhibitor is related to the La protein. We have deferred further characterization of the inhibitor until the nature of the shortened U6 transcript is more fully defined.
Post-transcriptional Shortening of the U6 snRNA in VitroTo
address directly whether the shorter U6 transcript was generated during
or after transcription, we took advantage of the P.3 inhibitor to
produce full-length U6 transcripts in the P.5 fraction. Transcripts
were purified from a high resolution denaturing gel and incubated in
the P.5 fraction for varying lengths of time. As shown in Fig.
2A, the transcript became progressively
shorter over a period of a few minutes. This result indicated that the processing activity was post-transcriptional. Similar experiments performed with other pol III- or pol II-derived transcripts yielded no
detectable processing, which confirms that the processing activity is
specific (data not shown). The issue of specificity for U6 is
explicitly addressed below.
In Fig. 2A, RNA lengths of 102 and 103 nucleotides predominated. We do not know why these transcripts are slightly larger than those shown in Fig. 1B, lane 3. The fact that the RNA produced in Fig. 1B was generated and available for processing over a course of 30 min, while the RNA in Fig. 2A was exposed to the processing activity for only a few minutes, might account for the difference.
To study the U6 processing reaction in more detail, large quantities of
purified U6 RNA were generated in vitro using T7 RNA polymerase. To fuse the T7 promoter to the U6 coding sequence, an
oligonucleotide containing the T7 promoter plus the 5-coding sequences
of U6 and an oligonucleotide containing the 3
-coding sequences were
used to amplify the U6 gene by PCR. The resulting DNA was purified and
incubated with T7 RNA polymerase to generate U6 RNA. In addition, the
processing activity was further purified by Mono Q chromatography. As
shown in Fig. 2B, this synthetic 106-nucleotide U6 RNA was rapidly
processed to 102 nucleotides.
With this experimental setup, we consistently observed a low level of shortening to 103 nucleotides, and a high level of shortening to 102 nucleotides, but no shorter. From day to day, low levels of contaminating nonspecific RNases in the system caused varying degrees of nonspecific degradation of the RNA. Thus quantitation of processing on an absolute scale was not feasible. However, as presented below, we were able to quantitate the fractional extent of processing.
U6 Nuclease Trims Four Nucleotides from the 3Because of earlier reports of in vivo shortening of
the U6 3 end (11, 14), we examined whether nucleotides were being removed from its 3
end. We reasoned that if three or four nucleotides were enzymatically removed from the 3
end of the wild type
106-nucleotide U6 RNA, then a 3
-truncated RNA that is missing the last
two Us should have only an additional one or two nucleotides removed. Indeed, as shown in Fig. 3, the wild type and
104-nucleotide U6 RNA, termed U6(1-104), were processed to the same
length. Therefore, the processing activity appears to be a nuclease
that trims three to four nucleotides from the 3
end of wild type U6
RNA. The results with U6(1-104) suggest that the nuclease removes
nucleotides up to a specified position, in this case predominately
nucleotide 102, as opposed to removing a fixed number of nucleotides
from the 3
end of any competent substrate RNA. That 105-, 104-, and 103-nucleotide intermediates are observed on the wild type U6 (e.g. see Figs. 1B, 2A, and 4)
suggests that the nuclease is a 3
,5
-exonuclease as opposed to a
site-specific endonuclease.
Kinetics of Processing
Having a kinetic assay in hand for monitoring the processing of the U6 RNA, we sought to obtain more information on the mechanism of processing. The rates of processing were measured at various concentrations of U6 nuclease (Fig. 4A). The relative level of processing was determined by the equation: processing (%) = 100P/(U + P), where P denotes the level of processed RNA and U denotes the level of unprocessed RNA. At the highest concentration of nuclease, the processing reaction possessed a t1/2 of ~4 s, suggesting that processing is relatively rapid. As expected, the rate of processing increased linearly with the amount of U6 nuclease added (Fig. 4B). However, with the lowest amount of U6 nuclease added, the reaction did not proceed to completion. A significant proportion of the U6 RNA was either not processed or only slowly processed. This type of behavior would be expected of an enzyme that slowly turns over. In other words, when there is more U6 nuclease than substrate, then the RNA is saturated and the processing reaction is rapid. When there is less U6 nuclease than RNA substrate, then to obtain processing of all the RNA, the nuclease must dissociate from the processed product and reassociate with unprocessed substrate. This turnover or product release appears to be inherently slow and overall rate-limiting.
Many nuclease reactions require magnesium. As shown in Fig.
5, the U6 nuclease also requires magnesium for activity.
In the presence of magnesium, the U6 RNA is completely processed within 40 s. In the absence of magnesium, no processing was observed over
a period of 60 s. When magnesium was added at 60 s,
processing was restored. Interestingly, when magnesium was added after
preincubation of RNA and nuclease, the kinetics of processing were very
slow. It is unlikely that the U6 nuclease was generally inactivated, since on longer incubations the reactions went to completion (data not
shown). Instead, the kinetics were reminiscent of the slow enzyme
turnover described in Fig. 4. One plausible interpretation of the data
in Fig. 5 is that the U6 nuclease binds U6 RNA in the absence of
magnesium but must dissociate from the RNA in order to chelate the
magnesium that is necessary for processing. We emphasize that these
mechanistic interpretations are tentative until more detailed studies
are performed to rigorously establish whether product release is
rate-limiting in multiple rounds of processing and whether magnesium
binding must proceed by an ordered mechanism.
Substrate Specificity of U6 Nuclease
The observed processing of the U6 RNA leads us to pose the following questions with regard to substrate specificity. 1) What features of the U6 RNA target it for processing and not other RNAs? 2) What features of the U6 RNA cause processing to terminate at nucleotide 102? 3) What role does secondary structure play? 4) Since the U6 RNA ends with four Us, is U6 nuclease uridylate-specific? While we cannot provide complete answers to all of these questions, we have begun to address them though mutagenesis of the U6 RNA.
Our first step in addressing these questions was to perform deletion
analyses on the U6 RNA. In Fig. 6A, time
courses of processing are presented for wild type U6, a 3 deletion
U6(1-76), and a 5
deletion U6(58-106). Above the wild type U6
reaction is shown a proposed secondary structure for the human U6 snRNA
as described by Epstein et al. (27). Above the two deletion
constructs are representations of the truncated RNAs. No effort was
made to determine their potential secondary structure; they serve only
to delineate the deletion end points. Processing was not detected for
either of these truncated RNAs, which confirms that the U6 nuclease is not a general nuclease but recognizes specific determinants on the U6
RNA. Since U6(1-104) was readily processed (Fig. 3), we can conclude
that an important sequence or structural element lies between
nucleotides 76 and 104. In addition, an important sequence or
structural element lies within the first 57 nucleotides of the U6
RNA.
As shown in Fig. 6A, the U6 RNA appears to have two major
hairpin structures. The four 3 Us that are processed lie at the base
of the large hairpin. We were curious as to whether the region complementary to the processed nucleotides was important for
processing. To this end, we compared the processing of two RNAs, one
termed U6(27-106), which was truncated from the 5
end up to the
hairpin and the other termed U6(33-106), which was truncated six
nucleotides into the hairpin. As shown in Fig. 6B,
U6(27-106) was readily processed while U6(33-106) was not processed.
This result indicated that the 5
end of an important sequence and/or
structural element for U6 RNA processing resides between nucleotides 27 and 33. From this data alone we cannot discern whether this region
provides important structural and/or sequence information.
To address the importance of secondary structure in the processed
region and characterize the sequence and length requirements in this
region, a battery of 5 and 3
mutants was generated. A summary of the
data is presented in Fig. 7. We point out the following
observations.
1) When one or three nucleotides were added to the end of U6 (mutants
107G and 107U3), efficient processing back to nucleotide 102 was observed. Thus, U6 nuclease possesses the necessary activity to
remove the added nontemplate 3 Us that are normally found on U6
in vivo. The fact that short 3
deletions and extensions were fully processed back to nucleotide 102 indicated that the nuclease
is not measuring four nucleotides. Instead it appears simply to stop at
position 102, which from the 3
end is the first position to contain an
A.
2) When the secondary structure at the 3 end was partially or fully
disrupted with one or more mutations between nucleotides 103 and 106 (mutants U3103C3,
U4103G4, U4103A4,
U105G, and U106G), processing back to nucleotide 102 was still
observed, although in some cases much less efficiently. Surprisingly,
some of these mutations included complete replacement of the four
terminal Us with either As or Gs. Thus, neither a stretch of Us nor
secondary structure at the 3
end of U6 appears to be essential for
processing. The observation that some of these mutations are
significantly depressed for processing, such as
U3103C3, suggests that this region does provide
some important sequence information.
3) U6 RNAs containing clustered mutations extending from nucleotide 28 to 30 (A328G3), which forms the upstream base of the major stem loop, were efficiently processed, demonstrating that this region provides neither sequence nor secondary structure information for processing. When secondary structure in this region was restored with GCs (mutant A328G3·U3103C3) instead of AUs, processing was relatively unaffected when compared with U3103C3.
4) Since nucleotide 102 was the first position from the 3 end in
U6(wild type) that contained an A, we initially wondered whether the
presence of an A signified a stop. However, mutant U6(U4103A4), which replaces the four terminal
Us with As, was efficiently processed back to position 102, thus
indicating that an A anywhere along the path of the nuclease does not
suffice to terminate processing. To examine what role A102 played, we mutated it to a U. The mutation caused two dramatic effects; first, U6(A102U) was poorly processed, indicating that A102 was important for
the overall level of processing, and second, U6(A102U) was processed
back to A100 and not to the expected nucleotide 102. Thus, it appears
that an A at nucleotide 102, but not at a site downstream of this
position, is important for terminating the processing. When A102 is
replaced with a U, U6 nuclease continues on through U102 and U101 and
finally terminates at A100. Apparently, an A at or just upstream of
nucleotide 102 signifies a stop for U6 nuclease.
5) Thus far deletion analyses have located the 5 end of an important
element between nucleotides 27 and 33. Mutations at positions 28, 29, and 30 indicate that these nucleotides are relatively unimportant. This
suggests that the 5
end of the important element lies between
nucleotides 31 and 33. Nucleotide U31 is paired with the important A102
in the proposed secondary structure. To address whether U31 was
important for processing, it was changed to an A (mutant U31A) and was
found to significantly depress processing. To address whether potential
loss of secondary structure at this position was inhibiting processing,
a complementary mutation was made (U31A·A102U). No processing above
that with each of the individual mutants was observed, which indicated
that secondary structure alone at this position was not sufficient to
support processing. Instead, it appears that, minimally, nucleotides 31 and 102 provide important sequence information for processing. Whether
secondary structure at this position is also important cannot be
addressed by the data. However, that U31 and A102 are both important
and are normally base paired would suggest that U6 nuclease is
recognizing this region in the context of secondary structure.
Interestingly, U31 is one of only three Us in the U6 RNA that is
post-translationally modified to pseudouridine (27). The significance
of this modification is not known.
6) We note that in the presence of high levels of U6 nuclease all 3
mutants were processed as well as wild type. Only at lower levels of
nuclease were differences in processing apparent. The concentration
dependence suggested that the 3
region might stabilize the binding of
U6 nuclease to U6 RNA.
Compiling the findings
presented here leads us to suggest an initial working model for
processing of the U6 3 terminus. Many details are still sketchy and
need to be worked out. This view is intended solely as a guide for
designing future experiments to refine the mechanism. The U6 nuclease
in complex with magnesium targets U6 through RNA interactions in the
vicinity of U31 and A102. These two nucleotides are base paired in the
proposed secondary structure of U6, when not complexed with U4. Other
unmapped regions of interactions are likely, possibly including some
interactions with the 3
-terminal U tract. U6 nuclease then
nonspecifically hydrolyzes the nearby 3
end of the RNA, one nucleotide
at a time until it reaches position 102 where it stops. Critical
interactions of the nuclease with A102 might prevent the nuclease from
hydrolyzing this nucleotide. In the absence of A at 102, the nuclease
selects the next upstream A, albeit inefficiently, and hydrolysis
proceeds up to that point. At the completion of the reaction, the
nuclease slowly dissociates from the processed RNA and is available to process other U6 RNAs. The U6 processing reaction can be regulated by a
distinct inhibitor located in the P.3 fraction.
It is unclear why the cell processes the 3 end of U6. At least in
Xenopus oocytes, there appears to be an excess of U6 snRNA (11). Much of it contains variable numbers of nontemplate Us on the 3
end and is not associated with the U4·U6 snRNP complex. Removal of
most of the terminal Us and formation of a 2
,3
-cyclic phosphate
appear to be prerequisites for incorporation into the snRNP complex
(11, 14). Since U4 snRNA hybridizes with the U6 snRNA through U6's
major stem loop, a major unwinding of the U6 stem loop would be
necessary to transit into the mature state. Unwinding of the U6 stem
loop and subsequent incorporation into the U4·U6 snRNP complex might
initiate at the 3
end of U6. If so, then the 3
end of U6 would be
expected to be a highly regulated region. Processing of the U6 3
end
may be one step along the pathway toward initiating its incorporation
into the U4·U6 snRNP.
We thank Timothy S. Fisher, Robert S. Carter, and Carmelata Chitikila for assistance in experiments and protein purification.