Worm Breeder's Gazette 13(4): 47 (October 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

C. elegans U2AF65

Tom Blumenthal, Kris Lea and Diego Zorio

Department of Molecular and Cellular Biology, University of California,
Berkeley, CA 94707, and Department of Biology, Indiana University,
Bloomington, IN 47405

     C. elegans introns are AU-rich, lack a polypyrimidine tract and
consensus branch point, and have highly conserved boundary sequences. 
The 5' splice site is the same as in other animals, but the 3' splice site has
the unusually long consensus, UUUCAG.  In other systems from yeast to
man, it has been shown that the pre-mRNA is brought into the spliceosome
by interaction of the 5' splice site with U1 snRNP.  However, in
trans-splicing there is no 5' splice site on the pre-mRNA and U1 is thought
not to be involved.  How then does splicing get started?  Perhaps a
component of the splicing machinery recognizes the UUUCAG.  One idea
for such a component is the required splicing protein, U2AF65, which
recognizes the polypyrimidine tract in mammals.  In order to begin to test
this idea, we have obtained cDNA clones that encode the worm homolog to
U2AF65.  The fly and mammalian U2AF65 were sufficiently similar to one
another to enable us to use PCR with worm cDNA.  We then selected nearly
full-length cDNA clones from Leilani Miller's embryonic cDNA library, and
sequenced several of these clones.  There were clones containing partial SL1,
SL2 and an SL2 variant at their 5' ends, indicating that U2AF65 is a
downstream gene in an operon.
        All previously analyzed U2AF65 proteins have similar structures. 
They have an RS domain thought to be involved in protein-protein
interactions, a region required for interaction with the small subunit,
U2AF35, and three RNA recognition motifs (RRMs).  So far, no organisms
have been found to have more than one form of this protein.  However, C.
elegans is an exception.  We identified three types of cDNA clones, which
would encode three U2AF65 isoforms.  Of the 11 clones analyzed, 7 are of
the typical animal type.  The other four clones contain inserts of about 300
bp within the second RRM.  The inserts contain a stop codon near the 3'
end, so translation would yield a U2AF65 lacking the last two RRMs, but
containing a novel sequence instead.  One clone contains a deletion of 297
bp, eliminating the U2AF35-interaction domain.  We have detected all three
RNAs by RT-PCR, as well as polypeptides of the size expected for the two
larger forms on a Western blot with Roland Kanaar's fly U2AF65 antibody.
        Thus these cDNAs can potentially encode three different forms of
U2AF65.  This is particularly intriguing in light of the fact that C.
elegans  has three kinds of splicing, unlike all other organisms in which
U2AF65 has previously been studied.  It has cis-splicing, trans-splicing
with SL1 near the 5' ends of pre-mRNAs, and trans-splicing with SL2 to
downstream genes in polycistronic pre-mRNAs.  It is tempting to speculate
that the three different U2AF65 polypeptides could be involved in
catalyzing the three kinds of splicing.  If so, we predict that the major
form is involved in cis-splicing, because it is the most abundant and has a
structure identical to the other animal U2AF65's.  The insert could be
specific for trans-splicing, and the deletion of the small subunit
interaction domain could be a specialization involved in SL2
trans-splicing, which might not require the small subunit, but might
require instead an interaction with the cleavage and polyadenylation

Acknowledgements:  This work was initiated during Tom's sabbatical in
Barbara Meyer's lab.  I wish to express my thanks to Barbara and to the
members of her lab (especially Mike Nonet and Dave Hsu) and to Roland
Kanaar from Don Rio's lab for all their help, advice, support and