314s Biochemical Society Transactions (1995) 23
Genomic organisation of plant U14 snoRNA genes.
DAVID J. LEADER, JANE F. SANDERS,
ANDREW TURNBULL-ROSS,
ROBBIE WAUGH and
JOHN W.S. BROWN.
Department of Cell and M o l e d a r Genetics, Scottish Crop
Research Institute, Invergowrie, Dundee DD2 5DA, Scotland,
UK.
The role of small nuclear RNAs in pre-messenger RNA
splicing is now well characterised in mammalian and yeast
systems. However, a second major class of small nuclear RNAs,
the small nucleolar RNAs (snoRNAs) involved in various aspects
of pre-ribosomal RNA @re-rRNA) processing and ribosome
maturation [ l ] is less well characterised. Whilst more than
twelve snoRNAs have been identified in yeast and over fifteen
in vertebrates, only two, U3 and U14, have homologues in both
systems. Furthermore, details of the functional properties of only
a few of these snoRNAs have been established [2,3]. For the
remainder, their function in rRNA metabolism is largely inferred
from nucleolar localisation, potential for base pairing with prerRNA and association with nucleolar proteins.
Recently there has been considerable interest in the discovery
that a number of animal snoRNA genes are encoded within the
introns of protein coding genes [2,3]. The first example of this
unusual genomic organisation was U14 genes which are located
in introns of the constitutively expressed hsc70 gene in a number
of animal species [4]. Expression of the U14 genes occurs via
a novel processing mechanism during which the U14 RNA is
excised from the intron [ S ] . There has been some discussion as
to whether pre-mRNA splicing is a prerequisite for snoRNA
processing. On the one hand, the snoRNA may be released from
the spliced intron following debranching, while in the other
model, splicing and snoRNA excision may be mutually exclusive
(5-81. In the latter case, initial endonucleolytic cleavage upstream
and downstream of the snoRNA would be required, whereas only
exonucleolytic activity would be needed to process the snoRNA
from the linear, debranched intron.
In plants, only two examples of snoRNAs U3 and 7-2NRP,
have been characterised and both are transcribed from promoters
similar to those of the spliceosomal snRNAs. As yet no
examples of intron-encoded snoRNAs have been demonstrated.
Here we report the isolation and characterisation of plant U14
genomic clones which show that, while some U14 genes occur
singly, others are organised in closely linked tandem gene
clusters and may be expressed via the processing of polycistronic
transcripts.
Primers were designed to highly conserved
sequences present in the U14 genes of yeast and vertebrates and
used in RT-PCR reactions with RNA from maize and potato to
generate putative partial U14 cDNA clones. Alignment of the
generated sequences allowed new primers to be designed to plant
specific U14 sequences for use in inverse PCR with plant
genomic DNA. Products generated by inverse PCR were
unexpectedly short and identical products were generated in
standard PCR indicating that they represented the amplification
of sequences between closely linked adjacent U14 genes. Probes
made from the cloned intergenic PCR (IgPCR) products were
used to isolate one potato and two maize genomic clones. One
of the maize genomic clones (ZmU14.1) contained four intact
U14 coding sequences and a short fragment of a fifth gene
within 760bp, whilst the second maize clone (ZmU14.4) and the
potato clone (StU14.1) contained single U14 genes. Neither the
flanking regions nor the intergenic regions between individuals
in the gene cluster or in the IgPCR generated fragments
contained the highly conserved plant UsnRNA promoter
elements. Similarly no other RNA polymerase I1 or Ill promoter
elements were found and the close proximity of the genes within
the gene cluster suggested that these genes may be expressed as
a single transcript from a common promoter.
The coding sequences of the cloned U14 genes show
extensive homology to, and regions of identity with, vertebrate
and yeast U14s. These include the highly conserved boxes C and
D, and regions A and B which have complementarity to
sequences in 18s-RNA [4]. Short inverted repeat sequences are
found at the 3' and 5' ends of the plant U14 genes and similar
sequences in vertebrates are suggested to be involved in the
processing of U14s from introns [ S ] .
RNase W1 protection analysis using gene specific probes
detected full length protected products within the range 118126nt. This variation in length could reflect variation in thc 3' or
5' termini of different U14 gene variants or that the termini of
plant U14 genes are not precisely defined. A similar population
of products was generated by primer extension using plant U14specific primers with maize or potato total RNA and the major
extension products mapped the 5' ends of the U14 transcripts
within the 5' inverted repeat sequences. Primers used originally
for Ig-PCR amplification of U14 genes were used in RT-PCR
with RNA from maize and potato and gave rise to multiple
products, while control reactions omitting reverse transcriptasc
gave no products. The cloning and sequencing of the Ig/RTPCR products confirmed the presence of 3' and 5' fragments of
adjacent genes and demonstrated that at least some of them were
identical to some of the Ig-PCR products and pairs of genes in
the gene cluser (ZmU14.1). Thus RT-PCR has detected a
number of transcripts containing adjacent U14 genes suggesting
that U14 gene clusters as seen in the genomic ZmU14.1 clone are
likely to be transcribed as polycistronic transcripts, and inferring
that individual U14s would be released by a processing pathway
which would require some form of endonucleolytic cleavage
between adjacent genes.
Analysis of the nucleotide composition of the sequences
flanking the U14 genes indicates that they are strongly W rich,
a property shared with plant intron sequences. However, the
sequencing of extensive regions upstream and downstream of thc
U14 genes has not identified strong candidates for exons.
Therefore, at present, we cannot determine whether plant U14
genes are encoded within introns or via transcription from a
novel promoter.
The research was supported by Gene Shears, Sydney,
Australia and the Scottish Agriculture and Fisheries Department.
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