Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick?

Abstract

Many neurogenetic disorders are caused by the mutation of ubiquitously expressed genes. One such disorder, spinal muscular atrophy, is caused by loss or mutation of the survival motor neuron1 gene (SMN1), leading to reduced SMN protein levels and a selective dysfunction of motor neurons. SMN, together with partner proteins, functions in the assembly of small nuclear ribonucleoproteins (snRNPs), which are important for pre-mRNA splicing. It has also been suggested that SMN might function in the assembly of other ribonucleoprotein complexes. Two hypotheses have been proposed to explain the molecular dysfunction that gives rise to spinal muscular atrophy (SMA) and its specificity to a particular group of neurons. The first hypothesis states that the loss of SMN's well-known function in snRNP assembly causes an alteration in the splicing of a specific gene (or genes). The second hypothesis proposes that SMN is crucial for the transport of mRNA in neurons and that disruption of this function results in SMA.

Figure 1. Function of SMN in snRNP assembly

Small nuclear ribonucleoproteins (snRNPs) are active in recognizing and removing introns from pre-mRNA in the nucleus. Each snRNP particle is composed of small nuclear RNA (snRNA) of approximately 150 nucleotides, several Sm proteins and a number of specific proteins that are unique for each snRNP. Survival motor neuron (SMN) functions in the cytoplasm to assemble Sm proteins onto the snRNAs to produce an active snRNP particle., , , . A) In the cytoplasm the 7 Sm proteins bind to the chloride conductance regulatory protein (pICln)., In vitro studies reveal that pICIn first binds the Sm proteins as two separate complexes: SmB, SmD3, and SmD1, SmD2. The latter subsequently binds SmE, SmF and SmG The protein arginine methyltrasferase (PRMT5 complex) and PRMT7 methylate the Sm proteins SmB, SmD1 and SmD3.,,, Sm proteins are released from pICln-PRMT5 complex and bind the SMN complex. B1) The SMN complex is composed of SMN, Gemins2-8 and unrip. SMN is shown in the figure as an oligomer as it has been shown to self-associate and it has been suggested that oligomerization is critical for SMN function (see text). The exact numbers of SMN monomers in a SMN complex is unknown (it has been suggested to be an octomer). The Gemins are shown as single units for simplicity as the exact stoichiometry of the SMN complex has not been determined. B2) snRNA is transcribed in the nucleus and then binds the export proteins phosphorylated adaptor for RNA export (PHAX), Cap-binding complex (CBC), exportin (Xpo1) and ras-related nuclear protein GTP (Ran), which transport it to the cytoplasm. In vertebrates, the snRNA is brought into the Sm protein-bound SMN complex by binding to Gemin5. C) The SMN complex places the Sm proteins onto the snRNA., The m⁷G cap of the snRNA is hypermethylated by trimethylguanosine sythetase 1 (TGS), allowing the SMN complex with the snRNA to bind snurportin and importin, which mediates transport of the SMN complex with an assembled snRNP into the nucleus. D) In the nucleus the SMN complex and snRNPs localize to the Cajal body and snRNPs undergo further maturation. Depending on the cell type and developmental stage, SMN can localize as a separate body adjacent to the Cajal body.– The figure is adapted from Pellizzoni.

Figure 2A. SMN1 and SMN2 genes: structure and splicing

The SMN1 and SMN2 have identical gene structure and are 99.9% identical at the sequence level. The essential difference between the two genes is a single nucleotide change in exon 7 (C or T as indicated). This single nucleotide change affects the splicing of the gene. Thus the majority of SMN transcripts from SMN2 lack exon 7 whereas those from SMN1 contain exon 7., , –, However, because SMN2 does produce some full-length SMN it can be viewed as a gene with reduced function but not loss of function. The loss of amino acids that are encoded by exon 7 results in the production of SMN protein with severely decreased oligomerization efficiency and stability., , , The SMN monomers are rapidly degraded. Thus, loss of SMN1 results in reduction of SMN levels in most tissues. The SMN oligomer is represented as an octomer based on gel filtration of SMN complexes formed in vitro Figure is taken from Butchbach and Burghes.

Figure 2B. Domains of SMN

A diagram of SMN showing the exons and domains. Exon 2B encodes a domain important in binding Gemin2 as well as self-association. Both exon 2A and 2B are conserved. The K domain is rich in lysine, the Tudor domain is in exon 3 and has homology to other Tudor domains. The Tudor domain binds Sm proteins. Exon 5 and part of exon 6 contain a proline rich domain that may influence profilin binding. The C-terminal domain of exon 6 contains the conserved YG box and is important for self-association.,

Figure 3. Mechanisms proposed to explain how reduced SMN levels cause SMA

According to one hypothesis, reduced SMN levels result in reduced assembly of Sm proteins onto snRNA. This unevenly alters the levels of specific endogenous snRNPs, such as those used to splice minor introns (particularly U11) from pre-mRNA., , It remains to be determined what the downstream target genes of the affected snRNPs are and how this specifically affects motor neuron function (indicated by a question mark (?)). One possibility is that the critical target gene is specific to motor neuron system. Alternatively, a function of critical importance to motor neurons could be disrupted. In addition, it has been suggested that reduced levels of β-actin mRNA or other mRNA occur at the axon tip or synapse due to SMN having a function in axon RNA transport, , at the growth cones of motor neurons cultured from SMA mice. It has been proposed that hRNPQ/R, and ZBP participate with SMN in this complex and that the reduced β-actin transport leads to alteration of calcium channel distribution at the axon terminal which in turn could affect neurotransmitter release (see text). Lsm proteins 1 and 4 have been found in axons in an RNP complex. We suggest that it is possible that reduced SMN levels affect the assembly of Lsm proteins required for axonal transport of mRNA, leading to reduced expression of specific genes at the synapse. However, a functional biochemical assay linking reduced SMN levels to an alteration in the formation of the required complex for transport of mRNA is lacking (indicated by ?). Whether other Lsm proteins, such as Lsm14, associate with this complex in neurons is not known. We have not indicated other potential or known SMN dependent assembly pathways, such as assembly of U7 snRNA, as it is not clear how alteration of this pathway would give rise to SMA. However, we cannot eliminate the possibility that other RNP assembly reactions are affected by reduced SMN levels. Lastly, it is possible to unite the two hypotheses where reduced snRNP assembly causes reduced splicing of a target gene that is critical for transport of mRNA to the motor neuron synapse.

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