Smaug variants in neural and non-neuronal cells

Abstract

Mammalian Smaug1/Samd4a is an mRNA regulator involved in synapse plasticity and additional non-neuronal functions. Here we analyzed the expression of Smaug1/Samd4a variants and Smaug2/Samd4b in primary hippocampal neurons and non-neuronal cell lines. We found that multiple Smaug proteins are present in several mammalian cell lines, including a canonical full length Smaug1, a Smaug1 variant that lacks the third exon, termed ΔEIII, and Smaug2, the product of a highly homologous gene. These three major isoforms are expressed differentially along neuron development and form cytosolic bodies when transfected in cell lines. By using luciferase reporters, we found that the ΔEIII isoform, which lacks 10 amino acids in the sterile α motif involved in RNA binding, shows a RNA-binding capacity and repressor activity comparable to that of the full length Smaug1. These observations are an important groundwork for molecular studies of the Smaug post-transcriptional pathway, which is relevant to neuron development, mitochondrial function and muscle physiology in health and disease.

Smaug membraneless organelles regulate mitochondrial function

“…Smaug orthologs are expressed in several mammalian cell lines (Fernandez-Alvarez et al, 2016) and as expected, we found that Smaug1 forms cytosolic puncta in U2OS cells (Figure 1A). Smaug1-bodies were present is most cells (70-80%, 5 independent experiments, N≥200 cells each) with highly variable numbers.…”

“…Additionally, a construct that lacks the first 53 amino acids and termed ΔSSR1 was not able to form cytosolic bodies (Figure 5C-D). Finally, the splicing variant ΔEIII also formed cytosolic bodies (Figure 5C-D) as reported before (Fernandez-Alvarez et al, 2016).…”

“…First, we analyzed whether the mitochondrial phenotype provoked by Smaug1 and Smaug2 double KD is efficiently rescued by transfection of Smaug1 (Figure 4). We found that this is the case and in addition, the splicing variant ΔEIII, which has similar RNA binding and repression capacity (Figure 1 and Fernandez-Alvarez et al, 2016), also rescued the mitochondrial phenotype (Figure 4). Altogether, these observations suggest that the three mammalian Smaug isoforms reported to date, Smaug1; Smaug1 ΔEIII and Smaug2 play a rather redundant role.…”

“…In contrast, we found that NDUFA10 mRNA was not significantly enriched and recovery values were close to those of PP1A mRNA (Figure 1D). Finally, Smaug1 ΔEIII, a natural splicing variant that forms similar cytosolic bodies (Fernandez-Alvarez et al, 2016) showed a similar profile. These results are comparable to observations reported in Drosophila, where SDHB mRNA is four-fold enriched in fly Smaug pull-downs and NDUFA10 mRNA is among the less-bounded transcripts (Chartier et al, 2015).…”

“…We have previously shown that transfected Smaug1 and Smaug2 form cytosolic bodies that colocalize (Fernandez-Alvarez et al, 2016) and we speculated that mammalian Smaug proteins may affect mitochondria activity. We investigated the effect of the double knockdown (KD) of Smaug1 and Smaug2, which were efficiently silenced by specific siRNAs (Figure 2A).…”