(Received for publication, April 3, 1997, and in revised form, June 25, 1997)
From Baylor College of Medicine, Department of Pharmacology, Houston, Texas 77030
Approximately 90% of human U6 small nuclear RNA
(snRNA) contains uridine cyclic phosphate (U>p) at its 3-end (Lund,
E., and Dahlberg, J. E. (1992) Science 255, 327-330).
We studied the formation of U>p at the 3
end of human U6 snRNA using
an in vitro system where uridylic acid residues are added
from UTP precursor and U>p is formed. Analysis of U6 snRNAs with
varying number of uridylic acid residues showed that each of these
species contains U>p where the phosphate originated from
-phosphate
of UTP precursor. The cyclic phosphate formation occurred on U6 snRNA
in extracts where essential spliceosomal snRNAs were specifically
degraded, thereby indicating that U>p formation is not coupled to
pre-mRNA splicing. A subpopulation of human signal recognition
particle and mitochondrial RNA processing RNAs isolated from HeLa cells
also contained cyclic phosphates at their 3
ends. These data suggest
that U>p in U6 snRNA is unlikely to be related to its participation in
splicing of pre-mRNAs. It appears that cyclic phosphate is an
intermediate product in the metabolism of these small RNAs.
Many small RNAs are known to participate in important cellular
functions. U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs)1 are required
co-factors for splicing of nuclear pre-mRNAs (1-4). RNaseP, U7
RNA, and many snRNAs are necessary for the site-specific cleavage of
pre-tRNA (5), histone pre-mRNA (6), and site-specific methylation
of pre-ribosomal RNAs (7, 8), respectively. SRP particles that contain
7SL RNA recognize the signal peptide of the secretory proteins and
participate in the secretion of these proteins (9). MRP RNA is found
mostly in the nucleolus and is required for the accurate 5 end
formation of the ribosomal 5.8 S RNA (10, 11). In addition, there are
many other small RNAs that are believed to play important roles in both
eukaryotic and prokaryotic cells (1, 12, 13).
Most RNAs undergo post-transcriptional modifications at their 3 ends.
These modifications include polyadenylation of eukaryotic mRNAs
(14) and -CCA addition/turnover on tRNAs (15). Most eukaryotic small
RNAs transcribed by RNA polymerase III, such as ribosomal 5 S, U6, SRP,
MRP, RNaseP, 7SK, and plant U3 RNA, terminate with 4-5 uridylic acid
residues. Although some of them, like 5 S RNAs, retain their 3
ends
and are found in the functional ribosomes with -UUUU-OH or -UUUUU-OH,
several others are processed at their 3
ends and contain sequences
slightly different from the original transcripts. Rinke and Steitz (16)
identified a small fraction (~10%) of the total U6 snRNAs that has
longer 3
ends with multiple uridylic acid residues and associates with La protein. Studies in our lab showed that the 3
end of U6 snRNA in
snRNPs specifically get labeled in vitro when
[
-32P]UTP is added to the cell extracts (17). This
3
-uridylation of U6 snRNA has also been studied by Hirai et
al. (18), Lund and Dahlberg (19), and Tazi et al. (20).
Lund and Dahlberg (19) showed that about 90% of the U6 snRNA in human
cells contains U>p at its 3
end, and Brunel and co-workers concluded
that this U>p formation is coupled to its involvement in splicing of
pre-mRNAs (20, 21).
In addition to U6 snRNA, many RNAs are known to contain 2,3
-cyclic
phosphate structures. The autolytic products of the hammerhead cleavage
of the satellite RNA of tobacco ringspot virus (22) and avocado
sunblotch viroid RNA (23, 24) contain 2
,3
-cyclic phosphates. Several
other autolytic motifs, such as hairpin, also cleave to give
2
,3
-cyclic phosphate (25). The cleavage of pre-tRNA by an
endonuclease results in a 5
half-molecule containing 2
,3
-cyclic
phosphate (26-28). The self-excision of a plant pre-tRNA also results
in RNAs containing 2
,3
-cyclic phosphate (29). Shumyatsky et
al. (30) analyzed Ehrlich ascites carcinoma (mouse) cell RNAs
fractionated on sucrose density gradients and analyzed poly(A)+ RNA isolated from light, intermediate, and heavy
fractions. Cyclic phosphates (pC>p and pU>p) were found in the
poly(A)+ fraction obtained from the intermediate fraction.
The synthesis of these cyclic phosphate-containing RNAs was inhibited
by low concentrations of
-amanitin, indicating that cyclic phosphate may be present at the 3
end of mRNAs. Nilsen and his colleagues studied the human pre-rRNA processing in vitro and found
2
,3
-cyclic phosphate cleavage intermediates (31). These data show
that the formation of cyclic phosphate in RNA is widespread in
nature.
In this study, we further characterized the formation of cyclic
phosphate at the 3 end of U6 snRNA. We also report that cyclic phosphate is present at the 3
end of other small RNAs. In addition, the U>p appears to be an intermediate after the removal of a
nucleoside from RNAs containing 3
-OH by an exonuclease.
Fine chemicals, including nucleoside
2,3
-cyclic phosphates, were obtained from Sigma.
[
-32P]ATP, [
-32P]CTP, and
[
-32P]UTP were obtained from Amersham Corp., and
[32P]orthophosphate was obtained from ICN.
For preparation of uniformly labeled RNAs, HeLa cells were incubated at 37 °C with [32P]orthophosphate for 16 h in phosphate-free medium (32). The 4-8 S RNA was prepared by centrifugation of the whole cell RNA on a sucrose density gradient and pooling the fractions corresponding to 4-8 S RNAs (33). The RNAs were fractionated and purified on a 10% denaturing polyacrylamide gel containing 7 M urea.
Hybrid SelectionDNA dot hybridizations were carried out as described by Kafatos et al. (34). 5 µg each of cloned DNA was immobilized on nitrocellulose discs and hybridized at 42 °C for 16 h with uniformly 32P-labeled HeLa 4-8 S RNAs. Hybridization solution contained 50% formamide, 80 mM Tris, pH 7.5, 600 mM NaCl, 4 mM EDTA, 1.5 × Denhardt's reagent, 0.01% SDS, and 5 µg/ml yeast tRNA. The filters were washed at 42 °C with 3 × SSC, 1.5 × SSC, and 0.5 × SSC containing 0.1% SDS. The hybridized RNAs were eluted with sterile water at 100 °C for 5 min., precipitated in presence of carrier yeast tRNA, and electrophoresed on denaturing polyacrylamide gels.
Digestion with Nuclease P1 and Other EnzymesThe RNAs were
digested with nuclease P1 at a 1:1500 (w/w) enzyme to substrate ratio
at 37 °C for 30 min. Usually, 30 µg of RNA was digested with 20 ng
of nuclease P1 in 25 µl of 10 mM acetate buffer, pH 5.0. The digests were either spotted on DEAE-cellulose paper and subjected
to electrophoresis or spotted on cellulose plate and subjected to
chromatography. Digestion with T1 RNase was done in 10 mM
Tris-HCl, pH 7.4, at a 1:10 (w/w) enzyme to substrate ratio at 37 °C
for 60 min. Labeled standard pU>p was prepared by labeling U>p with
polynucleotide kinase and [-32P]ATP.
The RNase H
digestion of U1 or U2 snRNAs was done according to Black et
al. (35). The oligonucleotides used in this study were
5-TGCCAGGTAAGTAT-3
(complementary to U1 snRNA nucleotide 1-14) and
5
-GAACAGATACTACACTTGA-3
(complementary to U2 snRNA nucleotide
27-45).
Electrophoresis on 57-80-cm-long DEAE-cellulose paper was carried out at 500 V for 4-5 h in Savant high voltage electrophoresis unit using 5% acetic acid/ammonium hydroxide buffer, pH 3.5. Chromatography on cellulose plates was performed as described by Silberklang et al. (36). The first dimension solvent was isobutyric acid/water/ammonium hydroxide (66:33:1, v/v/v), and the second dimension solvent was 0.1 M sodium phosphate buffer, pH 6.8/ammonium sulfate/n-propanol (100:60:2, v/w/v). In some cases, only the second dimension was performed for the separation of pU and pU>p.
As shown earlier by Lund
and Dahlberg (19), U6 snRNA from HeLa cells labeled in vivo
with [32P]orthophosphate contained pU>p (Fig.
1D). Incubation of HeLa cell
extract with [-32P]UTP resulted in rapid labeling of
U6 snRNA, which was the major labeled RNA (Fig. 1A,
lane 3). The labeled U6 snRNA was then digested with
different enzymes and subjected to chromatography. Digestion with
nuclease P1 yielded pU and pU>p (Fig. 1B, lane
3), and digestion with nuclease P1 followed by alkaline
phosphatase resulted in Pi and U>p (Fig. 1B,
lane 4). When the nuclease P1 digest was fractionated by
two-dimensional chromatography, radioactivity was observed in pU>p,
pU, and pUp (Fig. 1C). In several independent experiments,
the amount of pU>p and pUp were 10-30 and 1-2% of the total
radioactivity, respectively. The isolation and subsequent digestion of
pU>p with alkaline phosphatase yielded two labeled spots corresponding
to Pi and U>p (data not shown). These data show that some
of the U6 snRNA 3
ends formed in vitro contain U>p; in
addition, these data show that both the 5
- and 3
-phosphate residues
in pU>p originated from the labeled
-phosphate of the precursor
UTP.
Addition of [-32P]CTP or [
-32P]ATP to
HeLa cell extract resulted in labeling of tRNA 3
ends (Fig.
1A, lanes 1 and 2). Digestion of
labeled tRNA with nuclease P1 yielded only pC or pA (Fig.
1B, lanes 1 and 2), and no detectable
cyclic phosphate was found. These data show that formation of cyclic
phosphate is specific to U6 snRNA and does not occur in all cases where
nucleotides are added to the 3
end of RNAs.
Previous data showed that there are
multiple U6 snRNAs differing in the number of uridylic acid residues at
the 3 end (16, 37). When labeled U6 snRNA was fractionated on a short
polyacrylamide gel (Fig. 1A, lane 3), multiple U6
snRNA species did not separate. To facilitate separation of U6 snRNA
differing in number of uridylic acid residues at the 3
end, the U6
snRNA labeled in vitro (Fig. 1A, lane
3) was digested with T1 RNase to completion and then fractionated
by electrophoresis on a long 20% polyacrylamide gel (Fig.
2A, lane 2). A U6
snRNA T1 RNase digestion fragment corresponding to the 3
end with four
U residues was electrophoresed to serve as a standard (Fig.
2A, lane 1). The position of this fragment was
designated as 0, because this fragment with four uridylic acid residues
represents the U6 snRNA transcript as transcribed by RNA polymerase III
(37-39). Thirteen labeled bands were visualized with varying
intensities; these fragments correspond to seven additional U residues
to five deleted nucleotides when compared with U6 snRNA transcribed by
RNA polymerase III. These labeled RNA fragments were purified and
analyzed for the presence of cyclic phosphate. Both longer and shorter
U6 snRNA forms contained U>p (Fig. 2B). These data show
that the presence of U>p is not restricted to mature U6 snRNA with
4-5 uridylic acid residues.
Splicing Is Not Required for U>p Formation in U6 snRNA
Tazi
et al. (20) studied the formation of U>p at the 3 end of
U6 snRNA and concluded that U>p formation is coupled to an event
arising during splicing. Complementary oligonucleotide-directed RNase H
cleavage was employed to degrade U1 or U2 snRNAs, which are essential
for splicing of pre-mRNAs (35). Fig.
3A shows the analysis of
snRNAs after targeted degradation. There was no detectable intact U1
snRNA when oligonucleotide complementary to U1 snRNA was added (Fig.
3A, lane 2), and a similar result was obtained
when U2 snRNA was targeted (Fig. 3A, lane 3).
These data show that essential snRNAs were effectively degraded by this procedure.
Next, the U1 and U2 snRNA depleted extracts were used for 3 end
labeling of U6 snRNA in vitro (Fig. 3B). The
labeled U6 snRNAs from the control (Fig. 3B, lane
1) and snRNA-depleted extracts (Fig. 3B, lanes
2 and 3) were purified and analyzed for the presence of
U>p. There was U>p formation in each case, and the amounts of pU>p
in the control, U1, and U2 oligonucleotide-treated extracts were 29, 23, and 22% of total radioactivity, respectively (Fig. 3C,
lanes 1-3). These data show that the formation of U>p at
the 3
end of U6 snRNA does not require its participation in the
splicing of pre-mRNAs. Because the cyclic phosphate formation is
occurring in RNP particles, the marginal reduction in pU>p formation
upon U1 and U2 oligonucleotide treatment may be due to nonobligate linkage of cyclic phosphate formation to pre-mRNA splicing (see "Discussion").
HeLa cell 4-8 S RNA uniformly labeled with
[32P]orthophosphate was fractionated on a 50-cm-long
polyacrylamide gel and subjected to autoradiography (Fig.
4A). Two distinct bands of
roughly equal intensity were observed for SRP RNA (also known as 7SL
RNA), the RNA component of the SRP. The faster migrating band was
designated SRP-1 RNA, and the slower band was designated SRP-2 RNA.
Similar results were obtained with MRP RNA (also known as 7SM or 7-2
RNA). There were two bands of similar intensity where one was
designated MRP-1 RNA and the other was designated MRP-2 RNA.
Hybrid selection was carried out to confirm that these four distinct bands correspond to SRP or MRP RNAs. Human SRP DNA immobilized on nitrocellulose paper hybrid-selected both SRP-1 and SRP-2 RNAs (Fig. 4B, lane 2), and human MRP DNA hybrid-selected both MRP-1 and MRP-2 RNAs (Fig. 4B, lane 3). These data provide evidence for the identity of these RNAs. Under similar experimental conditions, U6 snRNA also fractionated into two major bands and several slower migrating bands of less intensity (Fig. 4B, lane 4), whereas 7SK RNA electrophoresed as one band (Fig. 4B, lane 1).
The SRP and MRP RNAs were purified, digested with nuclease P1, and
fractionated on a DEAE-cellulose paper. The SRP RNA is not known to
contain any modified nucleotides (33); therefore, only pppG and
mononucleotides were expected. However, in addition to pppG and
mononucleotides, another labeled compound comigrating with pU>p was
observed. This spot was more prominent in the SRP-1 RNA species than
SRP-2 RNA (Fig. 4C, lanes 3 and 4).
Similar results were obtained when MRP-1 and MRP-2 RNAs were analyzed
after nuclease P1 treatment. The faster migrating MRP-1 RNA species
contained significantly greater quantities of pU>p than MRP-2 RNA
(Fig. 4C, lanes 1 and 2). As controls,
human U6 snRNA and ribosomal 5 S RNA were also analyzed after nuclease
P1 digestion. As expected, a spot consistent with pU>p was observed in
U6 snRNA (Fig. 4C, lane 6), whereas there was no
detectable pU>p in 5 S RNA (Fig. 4C, lane 5).
The nuclease P1 digests of SRP-1 RNA and MRP-1 RNA were also subjected
to two-dimensional chromatography, and in both cases pU>p was observed
(Fig. 5, A and B).
These data show that in addition to U6 snRNA, a subset of SRP and MRP
RNAs also contain U>p.
The main findings of this study are: 1) U>p is not restricted to
mature U6 snRNA with 4-5 uridylic acid residues because longer and
shorter U6 snRNA species contain U>p; 2) Splicing is not required for
the formation of U>p at the 3 end of U6 snRNA. These data and data
from other investigators are consistent with a mechanism shown in Fig.
6 where trimming and addition of uridylic
acid residues to the 3
end of U6 snRNA is constantly occurring in a
dynamic equilibrium. In addition, this mechanism also implies
that U>p is an intermediate in the trimming process. The evidence for
this mechanism is as follows.
This mechanism (Fig. 6) predicts three populations of U6 snRNA: 3-OH,
3
-p, and 2
,3
>p. These products are formed in vitro and
have been identified by us in this study and also by other investigators (19, 20). Because [
-32P]UTP is the
source of the radioactivity and is added to the 3
-OH of U6 snRNA, the
necessary RNA substrates with 3
-OH should be present in
vitro. These substrates varied from 1 to 10 U residues (see Fig.
2A, lane 2). We found that the 3
-p in pU>p was
also labeled (Fig. 1B, lane 4); and this is
possible only by the addition of UMP and the subsequent removal of a
nucleoside. Therefore, these data provide evidence for a trimming
reaction in vitro involving deletion of a nucleoside. Though
the yield was rather low (1-2%), we also found pUp (Fig.
1C), which is a result of decyclization of U>p. Removal of
the 2
,3
-cyclization is required to make the 3
-OH group available for
the elongation reaction. The fact that U6 snRNAs-1 to -5 species were
found to be labeled in vitro (Fig. 2A, lane
2) shows that the U6 snRNAs where nucleotides are deleted are also
used for elongation in vitro. These data are also consistent with the observation of Lund and Dahlberg (19), who showed that the
terminal nucleotides of U6 snRNA are turning over in a
nontemplate-dependent manner.
The predicted intermediates with 3-OH, 3
-p, and 2
,3
>p are also
found in vivo. The identification of U6 snRNA with up to about 20 U residues (16) shows that necessary U6 snRNA substrates for
the addition of uridylic acid residues are available in
vivo. Because only RNAs with 3
-OH are substrates for the addition
of uridylic acids, different sizes of these U6 snRNAs must be
present in vivo. We also prepared cDNAs of U6 snRNA
obtained from immunoprecipitates of La antibodies and sequenced them.
Some of the resulting cloned cDNAs contained more as well as less
than 4-5 U residues as predicted from mature and predominant form of
human U6 snRNA.2 U6 snRNA
with 3
-p was also found in several species in vivo (19). In
yeast U6 snRNA, the 3
-p containing population was predominant. In
fruit fly, the 3
-OH, 3
-p, and 2
,3
>p populations were present at a
ratio of 1:1:1. Therefore, the U6 snRNA in vivo has three
populations with 3
-OH, 3
-p, and 2
,3
>p. These data are consistent
with the proposed mechanism shown in Fig. 6.
The enzymes involved in the trimming and elongation of the 3 end of U6
snRNA are not known. An enzymatic activity capable of forming cyclic
phosphate at the 3
end of RNAs has been characterized from HeLa cells
(40). However, the RNA substrate for this enzyme requires a
3
-phosphate, which is then converted into a cyclic phosphate. In the
case of U6 snRNA, the 3
-cyclic phosphate originates from the
5
-
-phosphate of the pppU used as the precursor. Therefore, enzymes
involved in the cyclic phosphate formation in U6 snRNA should be
different from this enzyme characterized by Filipowicz and Vincente
(40). There are many instances where an endonuclease or self-cleavage
of RNA results in RNA fragments with cyclic phosphate at the 3
ends
(see Introduction). In fact, any time the ester bond between
3
-phosphate and 5
-hydroxyl group in an RNA molecule is cleaved, the
2
,3
-cyclic phosphate is the initial product. However, the cyclic
phosphate formation in U6 snRNA 3
end could not be due to the action
of an endonuclease. Because our data identified a series of cyclic
phosphate-containing U6 snRNAs that differ by one nucleotide in length,
it appears that an exonuclease deletes a terminal nucleoside with a
cleavage of the ester bond between 3
-phosphate and 5
-hydroxyl
resulting in the formation of cyclic phosphate. Data from our lab (17)
and other groups (20) have shown that this 3
end modification is
occurring in RNP particles, and it is likely that the enzymes
responsible for 3
end modification in U6 snRNA recognize one or more
proteins associated with U6 snRNA. It would be necessary to
reconstitute U6 snRNPs in vitro for characterization and
eventual purification of the various enzymes involved in this
modification.
Tazi et al. (20) studied the U>p formation at the 3 end of
U6 snRNA and concluded that this formation is coupled to its involvement in splicing of pre-mRNA. Our data show that the
formation of spliceosome and U6 snRNA participation in the splicing are not required for the U>p formation at its 3
end. The 3
end turnover and U>p formation of U6 snRNA may be occurring within the spliceosome; however, our data indicate that it may be incidental to U6 snRNA being
present, and U>p formation is not coupled to its participation in
splicing. The observation of Lund and Dahlberg (19) that U6 snRNA in
some species has very little U>p at its 3
end is also indicative that
U>p is not likely to be functionally relevant or coupled to a step in
splicing of pre-mRNAs. In addition, the presence of U>p at the 3
ends of SRP and MRP RNAs, whose function is unrelated to splicing of
pre-mRNAs, also suggests that U>p formation is more likely to be
related to the metabolism of these small RNAs.
We thank M. Finley for providing HeLa cells and Y. Chen for valuable discussions.