Worm Breeder's Gazette 14(1): 90 (October 1, 1995)
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.
Laboratory of Genetics University of Wisconsin-Madison
Nonsense mutant mRNAs are generally less stable than WT
mRNAs. The seven smg genes are required for this "nonsense-mediated"
mRNA decay. In smg mutants, otherwise unstable nonsense mRNAs
accumulate to approximately WT levels. We have previously reported the
cloning of smg-5 (Anders and Anderson, 1995 Worm Meeting Abstracts) and
smg-7 (Cali and Anderson, 1995 Worm Meeting Abstracts). smg-5 encodes a
549 amino acid protein that is not similar to any protein sequence in
the public databases. smg-7 is predicted to encode a 53 kDa protein
with two notable features: (i.) three repeats of a conserved 34 amino
acid domain known as a tetratricopeptide repeat (TPR) and (ii.) a
negatively charged COOH-terminus. TPRs are predicted to form
amphipathic helices and are believed to mediate homo- and heteromeric
protein:protein interactions. Here we describe the characterization of
SMG-5- and SMG-7-specific antisera and provide evidence that these
proteins associate in vivo.
Polyclonal antisera to both SMG-5 and SMG-7 were generated in
rabbits using bacterially expressed fusion proteins as immunogen. Both
antisera specifically detect 0.1 ng of the appropriate fusion protein.
Antisera raised to SMG-5 fusion protein detect a 56 kDa protein in N2
but not smg-5(-) extracts by immunoprecipitation followed by Western
blotting. Antisera raised to SMG-7 fusion protein detect 54 and 57 kDa
proteins on Western blots of total worm protein. Several results
indicate the 54 kDa protein is encoded by smg-7. First, its apparent
molecular weight is quite close to that predicted for SMG-7. Second,
the abundance of the 54 kDa protein is reduced (but not eliminated) in
smg-7(r1131::Tc1) animals. smg-7(r1131::Tc1) animals accumulate
approximately 60% of WT levels of WT-sized smg-7 mRNA. Thus, it is not
surprising that this mutation does not eliminate SMG-7 synthesis. We
are currently investigating whether the 57 kDa protein is also encoded
by smg-7 (perhaps by an alternatively spliced smg-7 mRNA), and are
considering the possibility this protein represents a
post-translationally modified form of SMG-7.
The presence of a protein:protein interaction domain in SMG-7
suggests it might associate with other proteins, perhaps including the
products of some or all of the other smg genes. Therefore, we have
tested whether SMG-5 and SMG-7 associate in vivo as judged by
co-immunoprecipitation. Both SMG-5 and SMG-7 co-precipitate from
extracts when either antisera is used. The proteins are detected by
Western blot analysis of immunoprecipitates. Coprecipitation of SMG-7
with SMG-5-specific antisera requires WT SMG-5, as SMG-7 does not
coprecipitate from smg-5(-) extracts (although SMG-7 is present in
smg-5(-) extracts). Only the 54 kDa (not the 57 kDa) polypeptide
identified by anti-SMG-7 antisera co-precipitates with SMG-5. We
conclude that SMG-5 and SMG-7 physically interact as components of a
multi-protein complex. Work is in progress to determine if the two
proteins interact directly or indirectly, and if the integrity of the
complex is maintained in other smg mutants.