Structural basis for RNA recognition by a type II poly(A)-binding protein

We identified a functional domain (XlePABP2-TRP) of Xenopus laevis embryonic type II poly(A)-binding protein (XlePABP2). The NMR structure of XlePABP2-TRP revealed that the protein is a homodimer formed by the antiparallel association of β-strands from the single RNA recognition motif (RRM) domain of each subunit. In each subunit of the homodimer, the canonical RNA recognition site is occluded by a polyproline motif. Upon poly(A) binding, XlePABP2-TRP undergoes a dimer-monomer transition that removes the polyproline motif from the RNA recognition site and allows it to be replaced by the adenosine nucleotides of poly(A). Our results provide high-resolution structural information concerning type II PABPs and an example of a single RRM domain protein that transitions from a homodimer to a monomer upon RNA binding. These findings advance our understanding of RRM domain regulation, poly(A) recognition, and are relevant to understanding how type II PABPs function in mRNA processing and human disease

SMA MN cultures display a degenerative phenotype.

<p>(<b>A</b>) At 10 weeks of differentiation, both SMA patient-iPSC lines show a significant reduction of SMI-32+ MNs compared to both control iPSC lines. However, the Tuj1+ (βIII-tubulin) neuronal population is unaffected. These data are represented as mean ± SEM and are quantified in (<b>B</b> and <b>C</b>); the graphs are represented as percent positive TuJ1 or SMI-32 cells of the total Hoechst positive population. There was no significant difference in Tuj1 positive neuron numbers observed between the control and SMA cells at 3, 7 and 10 weeks of MN patterning. (<b>C</b>) There is a reduction of total cell body area and total number of processes in the SMA cell lines at late stages of differentiation compared to control iPSCs. (<b>D</b>) Meta-analysis of SMI-32 and TuJ1 counts confirms a significant reduction in SMI-32+ MNs in SMA cells. (<b>C</b> and <b>D</b>) Data are represented according to the longitudinal differentiation equation (<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039113#s4" target="_blank">Materials and Methods</a></i>) as mean ± SEM, n = 4 independent experiments. Scale bars  = 50 µm. * p<0.05, ** p<0.01.</p

Fas ligand over-expression in SMA MN cultures.

<p>(<b>A</b> and <b>B</b>) Expression of total membrane-bound Fas ligand was increased in 13iSMA cells after 6 week differentiation seen by (<b>A</b>) immunocytochemistry and (<b>B</b>) Western blot analysis. Data shown here are representative of n = 3 independent experiments.</p

Motor neuron (MN) differentiation from iPSCs.

<p>(<b>A</b>) Schematic representation of MN differentiation from iPSCs out to 10 weeks. (<b>B, C</b> and <b>D</b>) Differentiated human iPSCs are capable of developing into both (<b>B</b>) early, (<b>C</b>) intermediate, and (<b>D</b>) mature MN markers indicative of lineage restriction. (<b>D</b>) Detection of SMN protein in the nuclei (gems) and cytoplasm of choline acetyltransferase (ChAT) positive MNs derived from control iPSCs (83iCTR). Representative images for MN differentiation depicted here are from healthy control subject iPSCs (14iCTR or 83 iCTR). Scale bars: 50 µm.</p

Activation of caspase-8 in SMA MN cultures.

<p>(<b>A</b>) Western blot analysis of MN patterned SMA-iPSCs cell lysates at 8 weeks show reduced SMN and MN markers Islet-1 and HB9, but increased activation of caspase-8 compared to control iPSCs. There was no difference in expression levels of Bax, Bcl-2, and AIF. GAPDH was used as a loading control. (<b>B</b>) Immunocytochemistry detected an increase in cleaved caspase-8 in 13iSMA MN cultures<b>.</b> White arrows indicate doubled labeled HB9 positive MNs that are also positive for cleaved caspase-8. (<b>C</b>) An increase in caspase-8 activation in 13iSMA MNs was confirmed with the Caspase Glo-8 assay, measuring caspase-8 activity by release of luminescence upon activation of caspase-8 and a cleavage of a target peptide. Data are represented as mean ± SEM, n = 3 experiments.</p