Worm Breeder's Gazette 15(2): 45 (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.

Looking in C.elegans for the function of SMN, the Spinal Muscular Atrophy disease gene.

Solange Bertrandy1, Suzie Lefebvre1, Jo Anne Powell-Coffman2, Arnold Munnich1, Danielle Thierry-Mieg3

1 Hopital Necker-Enfants Malades, IFREM, Paris, France
2 Iowa State University, Ames, IA 50011
3 CRBM, CNRS, Montpellier, France (mail smn@kaa.crbm.cnrs-mop.fr)

We are grateful to the genome project for sequencing cosmid C41G7
(chromosome I, +3.7 cM), which contains an ortholog of the SMN
(Survival of Motor Neuron) gene. Mutations of the telomeric copy of
the duplicate SMN gene leads in human to the second most frequent
fatal autosomal recessive disease of childhood, spinal muscular
atrophy or SMA (Lefebvre et al., Cell 80, 155-165, 1995). The illness
is characterized by degeneration of motoneurons of the spinal cord. A
tight correlation between clinical severity and SMN protein level was
demonstrated in SMA patients and a strong protein expression was
observed in the spinal motor neurons from control individuals
(Lefebvre et al., Nature Genetics 16, 265-269, 1997). The function of
the SMN gene product is unknown. The SMN protein appears to form a
complex with spliceosomal snRNP proteins (Liu et al., Cell 90,
1013-1021, 1997) and to participate to spliceosomal snRNP biogenesis
(Fischer et al., Cell 90, 1023-1029, 1997). We wish to characterize
the nematode gene, its functions and regulations, in the hope that
this knowledge may help device therapeutic approaches for the 1/6000
unfortunate homozygotes.

No cDNA clone was available when we started the project a year ago. We
isolated, cloned and sequenced the full length cDNA. It measures 0.7
kb, does not appear to be transpliced and exactly matches the
genefinder prediction of the coding region (4 exons, 197 aa). We then
generated transgenic strains expressing a translational fusion of GFP
to the SMN genomic sequence, including the SMN promotor (0.53 kb of
upstream sequence, touching the nearest upstream gene, which encodes a
ncd-like kinesin). The SMN gene is fused to GFP-NLS (vector pPD95.70)
8 aa before the stop codon. Out of 44 Rol, only 2 transmitted. These
transgenics fluoresce at very low levels in most tissues, with two
dozen neurons (not motoneurons) a bit brighter than the other cells.

In the course of these experiments, we observed that injection of the
GFP fusion construct and the pRF4 Roller plasmid in the ratio of 50:
150 leads, 12 to 16 hours after the injection, to irreversible body
muscle paralysis of the injected worms. The injectees look healthy,
eat and lay between 60 and 140 eggs per hermaphrodite but move only
the head. The eggs laid will either die as embryos (85%), or hatch as
very short Lls, with a big head and an often knobbed tail (15%). We
are intrigued by the paralysis which directly affects the injected
animals and wonder if other cases of such "diffusible" effects were
ever observed ?

RNA interference experiments were also conducted. The injectees
generated a few small round eggs, some dead embryos and a proportion
of larvae that died as L2 or L3 and were either Unc (not paralysed,
but uncoordinated like the acetylcholine receptor mutants) and/or
strongly dumpy (like dpy-17). But the most striking failure was in the
differentiation of the germ line. Defects ranged from empty gonads (no
or few germ line cells, but normal somatic gonad), to Glp-l like (only
a few sperms), to small non growing ovocytes (vitellogenin accumulates
in the intestine, a few tiny round eggs may be laid, none survives). A
large number of animals also had misguided gonad(s), like Unc-5. 

Based on these phenotypic informations, we are selecting new alleles
in standard mutagenesis screens, using mnDfl11 as uncovering
deficiency and transgenics as wild type duplication. We are also
looking at previously identified mutants in the area.

In parallel, we started biochemical experiments. Rabbit antisera were
raised against three peptides, and we are currently using them in
immunoblots, and will try them soon in immunofluorescence.