A Role for Polyproline Motifs in the Spinal Muscular Atrophy Protein SMN

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of ␣-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.Spinal muscular atrophies (SMAs) 1 types I, II, and III are autosomal hereditary diseases of graded severity in which loss of motoneurons leads to paralysis and subsequent atrophy of skeletal muscles, and in the most severe Werdnig-Hoffmann type I form, to death in early infancy. The corresponding SMA disease genes have been mapped to human chromosome 5q (1). There are two genes in close vicinity, the telomeric SMN1 (or SMN T ) and the centromeric SMN2 (or SMN C ; 2). Although they have identical coding sequences for a 294-amino acid SMN polypeptide, pathogenic mutations were found solely in SMN1. Its gene product, the 40-kDa protein "survival motoneuron," SMN, is expressed ubiquitously, and its concentration is reduced drastically in the spinal cord of SMA patients (3, 4). There is evidence, mostly from a yeast two-hybrid screen and from a Xenopus oocyte model system, that the SMN protein is engaged in the assembly of spliceosomal U snRNPs in the cytoplasm (5-7). Recently, the function of SMN has been demonstrated by a dominant-negative mutant of SMN which inhibits pre-mRNA splicing by blocking the formation of a mature spliceosome (8).The severity of SMA has been correlated with a deficient oligomerization of mutated SMN proteins, and it has been hypothesized that the critical level of functional SMN oligomers in normal motoneurons may be controlled by SMNЈs binding to a motoneuron-specific factor (9). There are seven coding exo...

An essential SMN interacting protein (SIP1) is not involved in the phenotypic variability of spinal muscular atrophy (SMA)

“…Recently a strong interaction between SMN and a neuron specific profilin, PFNII, which is a microfilament-associated protein has been reported. 35 This also suggests that SMN may have other functions than in snRNP biogenesis and pre-mRNA splicing, and that its as yet unknown function may be responsible for the selective degeneration of the motor neurons in the spinal cord. Therefore, other SMN interacting proteins, like PFNII, or RNAs have to be analysed as potential SMA modifiers.…”

Profilin I is essential for cell survival and cell division in early mouse development

“…Part of the complication is that profilin interacts not only with actin but also with a large number of ligands such as Mena (13), VASP (14), dynamin I (15), gephyrin (16), and SMN (17), and that this ligand binding might be an important aspect of profilin function. The promiscuity of profilin for a plethora of signaling molecules provides links between a variety of cellular processes and actin remodeling, but it also complicates the interpretation of profilin's function in vivo.…”

Pathogenesis of proximal autosomal recessive spinal muscular atrophy

“…In addition to interactions with hnRNP-R and Q [69], SMN has also been shown to interact with profilin II, an actin-binding protein [31]. Profilin II is highly expressed in brain and spinal cord, where it localizes predominantly to anterior horn neurons [31]. The asymmetric SMN staining demonstrated in the germinative neuroepithelium further supported a role for SMN in neuronal migration and differentiation [23].…”

“…In addition to interactions with hnRNP-R and Q [69], SMN has also been shown to interact with profilin II, an actin-binding protein [31]. Profilin II is highly expressed in brain and spinal cord, where it localizes predominantly to anterior horn neurons [31].…”