Complete nucleotide sequence, genomic organization, and promoter analysis of the murine survival motor neuron gene (Smn).

Abstract

In humans, loss or mutation of the Survival Motor Neuron (SMN)gene is responsible for proximal spinal muscular atrophy (SMA),the second most common autosomal recessive disease of child-hood after cystic fibrosis. This lethal neuropathy affects 1/10,000live-born children. It is characterized by degeneration of thea-mo-tor neurons in the spinal cord, which causes proximal, symmetricallimb and trunk muscle weakness that progresses to paralysis. SinceSMA is clinically heterogeneous, patients are classified on thebasis of age at onset and disease severity (Munsat and Davies1992). Type I SMA children are the most severely affected. Theyhave onset of symptoms prior to 6 months, are never able to sit,and rarely live beyond 2 years of age. Type II and III SMA aremilder forms and show onset of symptoms between 6 months and17 years.Because of a 500-kb inverted duplication on Chr 5q13, theSMN gene is present in two copies designated centromeric SMN(cenSMN) and telomeric SMN (telSMN; Lefebvre et al. 1995).The genes span ∼30 kb, are highly homologous, and their ubiqui-tous transcripts differ by only four nucleotides, which are silentpolymorphisms (Lefebvre et al. 1995; Bu¨rglen et al. 1996; Braheet al. 1996; Hahnen and Wirth 1996; Chen et al. 1998). Individualslacking cenSMN are normal, whereas SMA patients have no de-tectable telSMN (>95%) or small intragenic mutations (Lefebvreet al. 1995; Bussaglia et al. 1995).Analysis of mRNA indicated that the human SMN genes arealternatively spliced and that telSMN produces three times theamount of full-length transcript (∼90%) compared with cenSMN(∼30%; Lefebvre et al. 1995; Gennarelli et al. 1995). SMN proteinis found in both the nucleus and cytoplasm and is present at highlevels in brain, kidney, and liver in normal tissues (Liu and Drey-fuss 1996; Coovert et al. 1997; Lefebvre et al. 1997; Francis et al.1998). There is only one copy of the SMN gene in rodents. Theabsence of alternative splicing in the mouse and rat suggests thatonly the product for the full-length SMN transcript is required fornormal development (DiDonato et al. 1997; Viollet et al. 1997;Battaglia et al. 1997). Recently, SMN protein was shown to beessential for spliceosomal snRNP biogenesis (Liu et al. 1997;Fischer et al. 1997) and, consistent with this housekeeping func-tion, complete loss of Smn leads to embryonic death prior to uter-ine implantation (Schrank et al. 1997). Despite these advances, itis not clear why defects in telSMN specifically affect motor neu-rons. One might speculate that motor neurons have a high require-ment for full-length SMN protein, which is not met when thetelSMN locus is disrupted. Alternatively, SMN may possess anadditional function specific to motor neurons, and loss of thisfunction causes SMA. To address these questions in vivo, it will benecessary to produce viable mice that harbor hypomorphic Smnalleles and/or Smn alleles that can be disrupted conditionally. Tofacilitate this process, we present the entire nucleotide sequence,genomic organization, a panel of unique probes that span the Smngene, as well as our gene targeting experience at the Smn locus.We have also analyzed the 58 region of Smn and show that itcontains a functional promoter.The Smn locus and flanking regions were completely se-quenced by a directed subcloning approach that was supplementedby PCR products amplified from 129/SvJ genomic DNA. Previ-ously characterized BAC clones, 411M1 and 227N6 (DiDonato etal. 1997), were used as source DNA to create BamHI, EcoRI, PstI,and Sa1I subclone libraries in pBSPT KS