Scientists agree that SMN works in the nucleus to help assemble the spliceosome, a protein complex that edits immature messenger RNAs (see ARF related news story on Zhang et al., 2008). But some suspect the protein also has a job in the cytoplasm (Rossoll and Bassell, 2009) since SMN-containing complexes turn up in motor neuron growth cones and processes (see ARF related news story on Fallini et al., 2010), and motor neurons from mouse models of spinal muscular atrophy (SMA) lack normal levels of some mRNAs at axon tips (Rossoll et al., 2003). In this study, the authors bolster the evidence for a cytoplasmic role by showing that SMN deficiency prevents HuD and mRNAs from reaching the ends of axons. Joint senior authors Wilfried Rossoll and Gary Bassell at Emory led the study.

HuD binds to the 3’ untranslated regions of certain mRNAs, including β-actin and tau, to stabilize them and regulate their localization and transport (Tiruchinapalli et al., 2008). The authors suspected that HuD and SMN might pair up because both are important for the targeting of growth-associated protein 43 (GAP43) to growth cones. Altered axon outgrowth is also a problem in SMA. Joint first authors Claudia Fallini, also at Emory, and Honglai Zhang at the Albert Einstein College of Medicine in the Bronx, New York, used pulldown experiments to show that SMN and HuD indeed interact in chick forebrain neurons, as well as in extracts from rat brains and spinal cords. The researchers also confirmed that HuD and SMN were an item in mouse primary motor neurons by employing a technique called bimolecular fluorescence complementation (BiFC), otherwise known as “split GFP” (Kerppola, 2008). “It is a very cool technique,” Fallini said. She cloned half of the gene for a fluorescent protein (yellow, in this case) onto a HuD construct, and the other half onto SMN. If the two proteins meet in the cell, the complementary pieces of the protein will come together and fluoresce. HuD and SMN did indeed hook up, producing a fluorescent signal that traveled up and down motor neuron axons. Another group of researchers also reported recently on the HuD-SMN partnership (Hubers et al., 2011).

Fallini and colleagues suspected SMN is required to dispatch HuD to axon ends. To test their idea, they used RNA interference to silence the SMN gene in cultured motor neurons. Via immunofluorescence, they observed that HuD signal in the axons dropped by one-quarter, while HuD levels in the cell body remained the same. Further, the researchers used fluorescence in-situ hybridization to label poly(A)-containing mRNAs in the SMN-silenced cells. The mRNA signal dimmed by more than half in axons, while again the cell body fluorescence was unchanged. “SMN has a function in the trafficking of RNA complexes along the axon,” Fallini concluded.

Could SMN-driven axonal transport be compromised in SMA? Mutations that cause the disease cluster in two parts of the SMN1 gene: a protein-protein interacting Tudor domain and a carboxyl-terminal tyrosine-glycine repeat section (YG box) involved in SMN homodimerization (Rossoll and Bassell, 2009). Fallini engineered these mutations into her SMN construct and coexpressed them with HuD in another split GFP experiment. A point mutation in the Tudor section diminished the fluorescent signal by a fifth compared to wild-type SMN, but the YG box substitution had no effect.

The Tudor mutant localized normally to axon tips—but was less able to bring HuD and associated components along for the ride. The YG mutant, in contrast, did not localize properly to axon tips. Either way, the outcome appears the same: mRNAs and proteins do not reach the ends of axons as they should. And for motor neurons, whose survival depends on translation of mRNA at axon tips, that is bad news. Thus, Rossoll and colleagues suggest that SMN mutations result in two distinct defects: one in nuclear mRNA splicing, and one in axonal mRNA transport. Next, the researchers hope to identify those RNAs and RNA-binding proteins that depend on SMN to reach the ends of axons.—Amber Dance.

Reference:
Fallini C, Zhang H, Su Y, Silani V, Singer RH, Rossoll W, Bassell GJ. The survival of motor neuron (SMN) protein interacts with the mRNA-binding protein HuD and regulates localization of poly(A) mRNA in primary motor neuron axons. J Neurosci. 2011 Mar 9;31(10):3914-25. Abstract