ABSTRACT
U14 is a small nucleolar RNA (snoRNA) required for early cleavages of eukaryotic
precursor rRNA. The U14 RNA from
Saccharomyces cerevisiae
is distinguished from its vertebrate homologues by the presence of a stem-loop domain that is essential for function. This element, known as the Y-domain, is located in the U14 sequence between two universal
sequences that base pair with 18S rRNA. Sequence data obtained for the U14
homologues from four additional phylogenetically distinct yeasts showed the Y-domain is not unique to
S
.
cerevisiae
. Comparison of the five Y-domain sequences revealed a common stem-loop structure with a conserved loop sequence that includes eight
invariant nucleotides. Conservation of these features suggests that the Y-domain is a recognition signal for an essential interaction. Several plant
U14 RNAs were found to contain similar structures, though with an unrelated
consensus sequence in the loop portion. The U14 gene from the most distantly
related yeast,
Schizosaccharomyces pombe
, was found to be active in
S
.
cerevisiae
, showing that Y-domain function is conserved and that U14 function can be provided by
variants in which the essential elements are embedded in dissimilar flanking
sequences. This last result suggests that U14 function may be determined solely
by the essential elements.
Several small nucleolar RNAs (snoRNAs) are now known to be involved in
processing of ribosomal RNA, and many others are expected to have roles in this
and other aspects of ribosome biogenesis (reviewed in
1
-
3
). The snoRNA populations in vertebrates and yeast are complex and a broad range
of potential functions is imagined, including folding of pre-rRNA as RNA chaperones, direct roles in cleavage, modification of rRNA
nucleotides and assembly of rRNP subunits (
1
,
4
,
5
).
U14 is a phylogenetically conserved snoRNA identified thus far in
Saccharomyces cerevisiae
, vertebrates and plants. Genetic depletion and mutation studies with
S
.
cerevisiae
showed that U14 is required for growth and that loss or inactivation of U14
disrupts cleavages which form a 20S precursor to 18S RNA (
6
,
7
). Comparison of U14 sequences revealed a common helix formed by the 5' and 3' ends, as well as four universal sequence elements, called boxes C
and D, and domains A and B. Functional mapping in yeast has shown each of the
conserved sequences and terminal stem to be essential (
8
,
9
). The box C and box D elements and terminal stem are required for U14
accumulation and are presumed to function in U14 maturation, most likely
processing, or incorporation of U14 into a stable snoRNP complex (
9
). The box D element in
Xenopus
U14 has been shown to be involved in formation of a 5' trimethylguanosine cap, although under unnatural conditions; this
phenomenon was observed for
in vitro
synthesized RNA containing a monomethylguanosine cap, following injection into
oocyte nuclei (
10
). This modification might not be relevant for
S
.
cerevisiae
U14 as the mature RNA lacks a 5' terminal cap. Box C has also been implicated in binding of the nucleolar
protein fibrillarin in studies of human U3, and both box elements influence the
metabolic stability of
Xenopus
U8 snoRNA (
10
-
12
). The two other universal sequences known as domains A and B (13 and 14 nt,
respectively) are complementary to conserved, well separated sequences in 18S
rRNA.
In vivo
genetic analysis has shown that U14 base pairs with the corresponding sequences
in rRNA (
13
).
The middle portion of the
S
.
cerevisiae
U14 sequence between domains A and B is considerably longer than the
corresponding segment in vertebrate U14 RNAs and accounts for nearly all of the
size difference between the larger
S
.
cerevisiae
RNA and the smaller vertebrate homologues (130 nt for
S
.
cerevisiae
versus 87 nt for mouse). Chemical and enzymatic probing data showed this
segment, called the Y-domain (yeast domain), to be highly structured in solution, forming an
extended stem-loop structure (
14
). The high degree of secondary folding suggests that the Y-domain might participate in either intra- or intermolecular interactions. While no information is yet
available about the function of this structure, deletion and substitution
mutations have shown that the Y-domain is essential for U14 activity in yeast. Variants with altered Y-domains accumulate normally, but the U14-dependent processing reactions do not occur (
8
, and W. Liang and M. Fournier, in preparation). Especially impressive was the
finding that the Y-domain endows mouse U14 with activity in yeast. Mouse U14, which differs
substantially from the yeast sequence in the non-conserved regions, is inactive in
S
.
cerevisiae
despite the fact that it accumulates at good levels. However, when the Y-domain was spliced into the appropriate region of the mouse U14 RNA, the
hybrid RNA could functionally substitute for yeast U14 (
15
). This finding showed that the Y-domain of
S
.
cerevisiae
can function in a different U14 context, and further suggested that U14
function may rely solely on the conserved elements.
With a view to gaining insights into the function of the Y-domain, we have analyzed partial or full sequences of several other yeast
U14 RNAs. The objectives of this study were: (i) to determine if the Y-domain is limited to
S
.
cerevisiae
and, (ii) if present in other yeasts, to analyze the phylogenetic sequences to obtain information about the structural requirements for Y-domain activity. The phylogenetically disparate yeasts
Kluyveromyces lactis
,
Candida albicans
,
Pichia pastoris
and
Schizosaccharomyces pombe
were all found to possess a Y-domain containing a non-conserved stem and a highly conserved consensus sequence in the
loop. Due to its sequence divergence, the gene encoding U14 was cloned from
S
.
pombe
. Despite differences in sequence and length,
S
.
pombe
U14 was able to functionally substitute for the
S
.
cerevisiae
U14, suggesting that U14 homologues are interchangeable in
S
.
cerevisiae
as long as they contain a Y-domain structure and the universal elements.
The
K
.
lactis
IFO 1090,
C
.
albicans
IFO 1385 and
S
.
pombe
975 yeast strains used for total DNA isolation were obtained from M. Fitzgerald-Hayes; the
P
.
pastoris
GS 115 strain was provided by T. Mason. All strains were grown on YEPD (1%
yeast extract, 2% peptone, 2% glucose) medium at 30oC. The
S
.
cerevisiae
galactose-dependent strain YS153 (MAT[alpha]
ura3-167
,
his3
,
trp1-1
,
HIS3::GAL1::U14
) (
8
) used for heterologous expression of
S
.
pombe
U14 RNA was grown on YNB (0.67% yeast nitrogen base) selective media containing
2% galactose or 5% glucose supplemented with necessary amino acids and nucleic
acid bases.
Escherichia coli
strain DH5[alpha] (
supE
44
lac
U169 ([Phi]
80 lacZ
-[Delta]M15)
hsdR
17
recA
1
endA
1
gyrA
96
thi
-1
relA
1) was used for most cloning procedures. Another
E
.
coli
strain, JM16 (
lac
U169
dam
3
rpsL
; M. J. Fournier, unpublished), was used when isolation of non-methylated plasmid DNA was required. Both bacterial strains were grown on
LB (0.5% yeast extract, 1% tryptone, 1% NaCl) medium supplemented with
ampicillin (50 [mu]g/ml), IPTG (isopropylthio-[beta]-d-galactoside, 40 [mu]g/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-[beta]-d-galactoside, 40 [mu]g/ml) when necessary. Bacterial strains were transformed with plasmids according to the protocol of D. Hanahan (
16
). The lithium acetate method (
17
) was used to introduce plasmid DNA into
S
.
cerevisiae
.
Plasmid DNA from
E
.
coli
was isolated by a boiling miniprep method (
18
). The procedure used to isolate yeast genomic DNA for PCR amplification,
Southern analysis and library construction is described by Rose
et al
. (
17
). Total yeast
RNA used for northern analysis was purified by a hot-phenol/glass bead method (
19
).
PCR amplification of total
K
.
lactis
,
P
.
pastoris
,
C
.
albicans
and
S
.
pombe
DNAs was carried out with Taq DNA-polymerase (BRL Life Technologies, Inc.) as follows: 35 cycles of 1 min at
95oC, 1 min at 25oC, 1 min at 70oC. The primers were complementary to the universal domain A and B
elements (which are complementary to 18S rRNA). The domain A primer includes
three additional nucleotides at the 3' end, and the domain B primer includes four additional nucleotides at the
5' end, corresponding to box D. The sequences of the primers were CATTCG[T
or C][A or G]CTTTCCAC (domain A side) and TCAGACATCCAAGGAAGG (domain B side).
After amplification, the PCR fragments were treated with T4 DNA-polymerase for 20 min at 15oC in the presence of 0.4 mM dNTPs and then subcloned into the
pBluescript IISK(-) vector. The sequences of the cloned fragments were determined using the
dsDNA Cycle Sequencing System Kit from BRL Life Technologies, Inc. and
pBluescript-specific primers SK and KS.
PCR fragments were labeled using [[alpha]-
32
P]dCTP (800 Ci/mmol; DuPont NEN) and a NEBlot Kit (New England Biolabs) and used
for northern analyses of total RNA isolated from the corresponding yeast
species.
Saccharomyces
cerevisiae
U14 was detected with the sequence-specific probe CGATGGGTTCGTAAGCGTACTCCTACCGTGG. The radiolabeled
S
.
pombe
fragment was also used for Southern analysis of
S
.
pombe
DNA. Heterologous expression of
S
.
pombe
U14 in
S
.
cerevisiae
was evaluated with radioactive
Cla
I DNA fragments (see below) labeled with the aid of random primers (NEBlot Kit).
All Southern and northern hybridization procedures were carried out essentially as described previously (
20
,
21
).
The restriction map of the
S
.
pombe
U14 genomic locus was developed from Southern analysis of four single and six
double restriction digests, after fractionation in a 1% agarose gel and
electrotransfer onto a nylon membrane. The enzymes used were
Eco
RI,
Hin
dIII,
Bam
HI and
Sal
I.
A
S
.
pombe
subgenomic library of 1500 colonies containing
Hin
dIII-
Sal
I DNA fragments of 1.1-1.3 kb (see also results) was prepared in a pUC18 vector essentially as
previously described (
21
) and screened using the radiolabeled PCR-fragment described above. Five identical plasmids were isolated from
colonies showing hybridization signals, and one was subjected to a detailed
restriction analysis. Fragments of 150-200 bp were subcloned into the pBluescript IISK(-) vector and sequenced as described above. Complete sequence
information was developed for both strands of the 1.2 kb
Hin
dIII-
Sal
I DNA fragment containing the U14 coding region. The sequence data were analyzed
with the program developed by the University of Wisconsin Genetics Computer
Group (
22
).
A first attempt to express
S
.
pombe
U14 RNA in
S
.
cerevisiae
used plasmid pRSPU14, which was constructed by inserting the 1.2 kb
Hin
dIII-
Sal
I genomic DNA fragment harboring the
S
.
pombe
U14 gene into yeast vector pRS316 (
23
) after digestion with
Hin
dIII and
Sal
I. A subsequent experiment was carried out with plasmids in which the
S
.
pombe
U14 coding sequence was inserted in place of the
S
.
cerevisiae
U14 segment in the context of a fragment of
S
.
cerevisiae
DNA. To prepare these plasmids, a 1.2 kb
Cla
I fragment containing
S
.
cerevisiae
genomic DNA with coding sequences for snR190 and U14 RNAs was isolated from
plasmid pJZ45 (
8
) and inserted into the
Cla
I-site of the vector pRS316 in both orientations (plasmids pCer1 and pCer2)
and separately into the
Cla
I-site of the vector pBluescript IISK(-) to produce plasmid pBCer. A
Bcl
I-
Bst
1107I fragment (130 bp) containing the coding region for
S
.
cerevisiae
U14 was replaced in plasmid pBCer with the genomic DNA fragment
Bcl
I-
Pac
I (145 bp) containing
S
.
pombe
U14, to generate plasmid pBPom; the DNA fragment ends formed by
Pac
I were blunted using T4 DNA polymerase prior to cloning. In the final step, the
Cla
I fragment containing the
S
.
pombe
DNA was cloned from the pBPom plasmid into the pRS316 vector in both
orientations to produce the plasmids pPom1 and pPom2.
To determine if the Y-domain occurs in other yeasts, we designed oligonucleotides corresponding
to the phylogenetically stable domains A and B and several adjacent conserved
nucleotides and used these for PCR amplification of total DNAs from
K
.
lactis
,
P
.
pastoris
,
C
.
albicans
and
S
.
pombe
. A single band product of ~80-90 bp was obtained in each case, and a positive control reaction
with
S
.
cerevisiae
DNA gave a product of similar size (data not shown). The PCR fragments were
radiolabeled and used to probe northern blots of total RNA from the homologous
yeast species. Strong signals were identified in the small RNA region in each
case, ranging in size from ~110 to ~130
nt. This compares with ~130 nt for the
S
.
cerevisiae
RNA, detected with a homologous probe (Fig.
1
A).
All the new sequences contain inverted repeats of 6-8 nt which can be arranged into secondary structures analogous to the
stem of the
S
.
cerevisiae
Y-domain (Fig.
1
C). The stems vary in length and differ in their nucleotide content;
nevertheless, the base pairing is always preserved. The content and sizes of
the loops are also variable, however, within an 11
nt core region of the loop, there is considerable homology. Eight of these
nucleotides are fully conserved, including the contiguous stretch GAACC. Two
additional nucleotides in this segment are identical in four of the five yeast
species, permitting a consensus sequence to be derived (Fig.
1
C). This high degree of sequence relatedness suggests that Y-domain function is conserved in all cases and that the conserved
nucleotides in particular are important for this function. Structure probing of
protein-free
S
.
cerevisiae
U14 RNA supported the existence of an additional stem element in the Y-domain involving several nucleotides in the consensus loop region (
14
). The potential to form the smaller helix is also present in
K
.
lactis
and
P
.
pastoris
RNAs, but is difficult to formulate for the U14 species from
C
.
albicans
and
S
.
pombe
.
The amplified U14 segment from
S
.
pombe
had the least resemblance to the corresponding region in
S
.
cerevisiae
U14, containing the shortest stem (6 bp), the smallest loop (14 nt), and the
shortest distance between the stem and domain B (16 nt). It was therefore of
interest to characterize the entire U14 sequence from this yeast and to assess
its function in
S
.
cerevisiae
. A detailed Southern analysis of
S
.
pombe
DNA, using the homologous PCR fragment as probe, showed the U14 gene to be
present in a single copy in the genome (not shown), and allowed us to make a
restriction map spanning almost 18 kb of this locus (see Materials and Methods,
Fig.
2
A). Using this map, a subgenomic library was constructed from
S
.
pombe
DNA containing
Hin
dIII
-Sal
I fragments ranging in size from 1.1 to 1.3 kb. The library was screened with
the radiolabeled
S
.
pombe
PCR fragment, and a 1.2 kb
Hin
dIII-
Sal
I fragment was cloned. The complete sequence of this fragment was determined
(GenBank accession number U29583). The
S
.
pombe
U14 coding region of ~110 nt was located based on its homology with
S
.
cerevisiae
U14. The approximate ends of the coding region were defined by comparison with
other U14 RNAs including that of
S
.
cerevisiae
, and estimation of the RNA size (Fig.
2
B).
Unlike the U14 locus in
S
.
cerevisiae
which contains a second snRNA (snR190) only 65-70 nt upstream of U14, the cloned 1.2 kb
S
.
pombe
fragment does not appear to encode any other small RNA. The sequence shows no
homology with snR190, and the fragment detects only U14 in a northern analysis
of
S
.
pombe
total RNA (data not shown).
Since all known vertebrate U14 RNAs, in contrast to
S
.
cerevisiae
U14, are encoded within introns of protein genes (
1
,
24
), we analyzed the 1.2 kb fragment containing the
S
.
pombe
U14 sequence for this possibility. Sequences matching the canonical intron
junction sequences (5' splice site, G/GTAWGT, and 3' splice site TAG, where W = A or T;
25
) could be found framing the U14 coding sequence, and sequences adhering to the
branchpoint consensus sequence (TRCTAAC, where R = A or G;
25
) were found between the junction-like sequences. However, no reasonably long open reading frames could be
identified outside the candidate junction sites, arguing that
S
.
pombe
U14 is not encoded in an intron, but is derived from an independent
transcription unit. This interpretation is supported by the presence of a TATA
box consensus sequence ~65 bp upstream of the coding region.
Schizosaccharomyces pombe
U14 contains, in addition to the Y-domain, all of the conserved features of the other U14 RNAs, i.e., domains
A and B, boxes C and D and a putative terminal stem (Fig.
3
A). Alignment of the
S
.
cerevisiae
and
S
.
pombe
U14 RNA sequences showed an overall identity level of
72% when the additional nucleotides in the
S
.
cerevisiae
homolog are excluded. Most of the signature elements, specifically boxes C and D
and domain A, are identical. Domain B of
S
.
pombe
is one nucleotide longer and contains one nucleotide difference. The
complementarity of domain A to
S
.
pombe
18S rRNA is perfect. Domain B of
S
.
cerevisiae
has one mismatch with its complementary element in 18S RNA. Complementarity is
perfect for
S
.
pombe
at this particular position, with the sequence difference located in U14 rather
than 18S RNA. However, a mismatch also occurs because of the extra nucleotide
contained within domain B of
S
.
pombe
U14. As for
S
.
cerevisiae
U14, the coding sequence in
S
.
pombe
is flanked by inverted repeats so that the potential to form an extended helix
in U14 precursors is maintained, although with different sequences. The
S
.
pombe
U14 RNA is 20 nt shorter than the
S
.
cerevisiae
homologue. While the differences in length are clustered throughout the
molecule, ~40% of this difference (8 nt) occurs in the Y-domain. The remaining differences occur in the non-conserved regions between the various signature elements.
To determine if
S
.
pombe
U14 can functionally substitute for
S
.
cerevisiae
U14, we first tried to express the
S
.
pombe
U14 gene in
S
.
cerevisiae
in the context of the original
S
.
pombe
genomic fragment (
Hin
dIII-
Sal
I, Fig.
2
A) cloned into a
S
.
cerevisiae
shuttle vector. This construct supported very limited growth of a galactose-dependent
S
.
cerevisiae
test strain on glucose medium, and a northern analysis with a
S
.
pombe
U14 probe did not give a readily detectable RNA signal (not shown). We note
that growth was screened on glucose medium, whereas RNA was prepared from cells
cultured on galactose in order that both positive and negative controls could
be obtained. It is possible that
S
.
pombe
RNA was expressed at a low level in glucose, as the cells would be under
greater selective pressure in this condition.
To avoid the possibility that the
S
.
pombe
transcription signals are poorly utilized in
S
.
cerevisiae
, we expressed the
S
.
pombe
U14 gene under control of the
S
.
cerevisiae
U14 promoter. U14 in
S
.
cerevisiae
is believed to be co-transcribed with the upstream snR190 and both snR190 and U14 are produced
by post-transcriptional processing (
1
,
6
,
14
).
To achieve expression, the
S
.
cerevisiae
U14 DNA was precisely excised and replaced with the corresponding region of
S
.
pombe
U14 DNA (Fig.
4
A).
Cla
I restriction fragments containing homologous and heterologous U14 segments were
inserted into yeast shuttle vector pRS316 in both orientations, to assess the
potential contributions of plasmid promoters. The resulting plasmids were
transformed along with a pRS316 vector into
S
.
cerevisiae
test strain YS153. This haploid strain contains a chromosomal copy of the U14
gene under GAL1 promoter control. The activity of the plasmid-encoded U14 RNAs was then tested by culturing on glucose-containing medium, which represses expression of the wild-type chromosomal U14 gene (
8
).
Characterization of conserved snoRNA elements is still at an early stage.
However, it is already clear that these elements fall into different classes,
based on phylogenetic conservation and presence in different snoRNA types.
Prior to this work two distinct classes were evident. One consisted of elements
conserved in several different snoRNAs in a wide range of organisms, from lower
to higher eukaryotes. The box C and box D elements are in this class, as they
are found not only in the U14 RNAs, but many other snoRNAs as well (
1
). The second class is defined by elements conserved among homologues of a
particular snoRNA, but absent thus far from other snoRNAs. The domain A and B
elements in U14 are in this class, as are the functionally undefined box A and
B elements common to all U3 snoRNAs.
A few elements, such as the stem and loop-IV domain of
S
.
cerevisiae
U3, which is absent from
S
.
pombe
U3 and from U3 homologues from non-yeast sources (
26
,
27
), fall into a class of organism-specific structures; the evolutionary importance of such elements is
unknown. Prior to this study the Y-domain also fell into this category, as it had only been observed in
S
.
cerevisiae
. Its presence in four other yeasts demonstrates that this element is not
organism-specific, and defines a novel category of elements, those that are
specific for a group of organisms.
Comparison of the Y-domain sequences from the five disparate yeasts yielded a simple consensus
structure that will be valuable for future structure-function studies. This structure consists of a stem-loop domain with a conserved sequence in the loop region (Fig.
1
C). The individual stems vary in length and sequence but sequence
complementarity is preserved, arguing that base pairing is essential for
function, perhaps for structural stability. The size and composition of the
loop segments also vary; however, there is considerable homology within an 11
nt region of the loop, permitting a consensus sequence to be derived. Eight of
these nucleotides are identical in all yeasts, including the contiguous stretch
GAACC.
Our finding that
S
.
pombe
U14 can function in
S
.
cerevisiae
indicates that this homologue contains all the structural features required for
activity, despite size, sequence and folding differences in the Y-domain and different flanking sequences surrounding the other essential
elements.
Schizosaccharomyces pombe
U14 is 20 nt shorter than its
S
.
cerevisiae
counterpart. The fact that it is active in
S
.
cerevisiae
indicates that the additional 20 nt in
S
.
cerevisiae
U14 are not essential for function. Eight of the 20 extra nucleotides are
within the Y-domain region, indicating that neither the size of the loop nor length of
the stem are critical for function, as implied by the consensus structure. The
remaining 12 nt fall in other non-conserved regions, providing additional evidence that function is
determined by the universal elements.
Our earlier functional analysis of mouse U14 in
S
.
cerevisiae
showed that the mammalian snoRNA can be expressed and is stable in yeast, but
does not provide the function required for rRNA processing (
15
). Activity was rescued, however, by insertion of the
S
.
cerevisiae
Y-domain in the same relative position it occupies in its normal host
snoRNA. Taken together, the results obtained with the
S
.
pombe
and the hybrid mouse U14 RNAs argue that the essential elements are able to
mediate their activities in an otherwise flexible molecule that varies in size
and sequence. The unrelated linking segments might only provide connector
function.
Since this work was completed sequences have become available for U14 RNA from
three plants, i.e., from maize (four species), potato and
Arabidopsis thaliana
. The maize and potato sequences came from direct
analyses (
28
), whereas that for
Arabidopsis
, which is partial, was identified by computer screening of the GenBank genomic
database (
28
,
29
). The identity of the
Arabidopsis
sequence is based on the presence of the signature elements and >95% identity
with an 80 nt segment of maize DNA. The plant sequences (~120 nt) are longer than the vertebrate homologues, but similar in size to
the yeast RNAs. Each contains the universal U14 sequence elements and the box C-box D-terminal stem motif (
28
). As with the yeast and vertebrate U14 homologues, considerable variation
occurs in the `non-conserved' sequences of the plant RNAs.
We have examined the folding potential of the plant sequences and, to our
surprise, determined that each contains a segment located between conserved
domains A and B that has the potential to form a stem-loop structure (Fig.
5
). Like the Y-domain, the stem elements of the plant domains (all 8 bp) vary in
sequence, but maintain complementarity. The loop segments contain 12-14 nt that display considerable homology, with nine of the nucleotides
being fully conserved in all three sequences. However, the consensus sequence
that can be derived,
C C - - Y G C C R G G C U,
exhibits no obvious relationship to that derived for the five yeast species
(A M G A A C C Y - A U).
The vital nature of the Y-domain was shown in previous deletion analyses of
S
.
cerevisiae
U14 (
8
). Our present phylogenetic comparison establishes the importance of the Y-domain structure and further implicates conserved nucleotides in the loop
sequence in carrying out its function. The conserved nucleotides can be
imagined to function in RNA base pairing or recognition by a protein. Base
pairing could be intramolecular, involving interaction with another region of
U14, or intermolecular, involving interaction with a different RNA, for
example, pre-rRNA or another snoRNA. Intramolecular base pairs can be predicted in some
cases, but these are short (<5
nt) and are not conserved in
S
.
pombe
(not shown). Interactions with other RNAs or protein are attractive
possibilities. This could be important for many aspects of U14 function,
including assembly or transport of the U14 snoRNP, or interaction of this
complex with components of the pre-rRNA folding and processing machinery.
The dissimilarity between the loop consensus sequences for yeast and plants
argues that the two domains have different recognition and interaction
properties, and perhaps even different functions. No functional information is
yet available for the Y-like domains in plant U14 RNAs. In time it will be interesting to test the
activity of this and the other plant U14 segments in
S
.
cerevisiae
, but this will be done in the context of higher resolution structure and
function studies of the
S
.
cerevisiae
elements.
Regardless of whether plants contain a functional homologue of the Y-domain, the question remains as to why this element is absent in the U14
RNAs of vertebrates. It is possible that this function is provided by other
elements in U14, but the inability of mouse U14 to function in yeast argues
that this is not the case, or that the yeast and animal elements are not
interchangeable. Folding models of mouse U14 do not reveal any candidate
sequence and structure that might specify this function (data not shown). It is
more likely that the function of the Y-domain in vertebrates is carried out by some other
trans
-acting molecule or is simply absent altogether. Identifying the role of
the Y-domain and characterizing its mechanism of action are important goals for
the future.
We thank Molly Fitzgerald-Hayes and Thomas Mason for providing yeast strains, and John Brown for
sharing plant U14 sequence data and helpful discussion. We appreciate the
assistance with computer analyses provided by Jingwei Ni and discusssions about
Y-domain functions with Wen-Qing Liang, both in our laboratory. We also thank Elizabeth Furter-Graves for expert editorial help. This research was supported
by NIH grant GM-19351.
REFERENCES
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