Worm Breeder's Gazette 15(2): 43 (February 1, 1998)

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.

SMG-2 is a phosphoprotein

Michelle Page, Philip Anderson

Laboratory of Genetics, University of Wisconsin-Madison, Madison WI 53706

Nonsense-mediated mRNA decay is a surveillance system that rapidly
degrades messenger RNAs containing premature stop codons.  
Nonsense-mediated mRNA decay has been observed in many eukaryotes
including yeast, Drosophila, humans and plants.  This nonessential
system of decay has been characterized genetically in C.elegans. 
Function of the seven smg genes is required for nonsense-mediated mRNA
decay.   Mutations in any of these genes abolish NMD, and nonsense
mutant mRNAs accumulate to wild-type levels. 

smg-2 encodes a protein containing a  probable C6 zinc finger domain,
and a type I RNA helicase domain with a nucleotide-binding motif.   We
raised antibodies against the amino terminal portion of a His-tagged
SMG-2, fusion protein.   These antibodies detect, on a western blot, a
single band of the expected 120 Kd in N2 animals.    No signal is
detected in smg-2 null mutants.   The same single 120Kd band is found in
smg-1, smg-3 and smg-4 mutants.  In smg-5, smg-6 and smg-7 mutants,
however, a more slowly migrating SMG-2 isoform is present in addition to
the abundant 120Kd isoform.   When smg-5 extracts are treated with
lambda phosphatase, the more slowly migrating isoform is no longer
detected, demonstrating that SMG-2 is a phosphoprotein and that the
phosphorylated form accumulates in smg-5, smg-6 and smg-7 mutants.  We
believe that phosphorylated SMG-2 is too transient to detect in N2

We have tested which smg genes are required for SMG-2 phosphorylation by
double-mutant analysis.  A smg mutant that accumulates the
phosphorylated form of SMG-2 can be combined with a second smg mutation.
If no phosphorylated form is detected in the double mutant, the second
smg gene is required for SMG-2 phosphorylation.  From this double mutant
analysis, smg-1, smg-3 and smg-4 are required for phosphorylation of
SMG-2.  SMG-1 encodes a putative PI-3/protein kinase domain.  We are
currently testing whether SMG-1 interacts with SMG-2 and if a
smg-1-dependent kinase activity coimmunoprecipates with SMG-2.

The analysis of SMG-2 in smg single and double mutants divides the smg
genes into two functional classes, those required to phosphorylate SMG-2
and those required for normal dephosphorylation of  SMG-2.  This is the
first grouping of genes involved in NMD and it is hoped this will
provide a framework on which to build a biochemical pathway.