Journal: Nature neuroscience

Article Title: Impaired neurogenesis alters brain biomechanics in a neuroprogenitor-based genetic subtype of congenital hydrocephalus

doi: 10.1038/s41593-022-01043-3

Figure Lengend Snippet: a, The TRIM71 polypeptide domain schematic shows clustering of all mutations in the RNA-binding NHL domain. b, TRIM71 immunostaining in the E9.5 forebrain neuroepithelia of WT and Trim71R595H/R595H. c, TRIM71 immunoblot in WT, Trim71R595H/+ and Trim71R595H/R595H mESCs. d, Immunoblots showing ubiquitin and SHCBP1 signals of SHCBP1-IP from Trim71fl/fl, Trim71-KO, WT and Trim71R595H/R595H mESCs differentiated with RA. SHCBP1 showed two bands; the upper band coincides with the ubiquitin signal. e, Quantitation of the SHCBP1 bands from SHCBP1-IP. Top SHCBP1 was normalized to total immunoprecipitated SHCBP1 (top + bottom bands). Trim71 KO and Trim71R595H/R595H values were then normalized to their respective controls. f, Autoradiograph of PAR-CLIP samples from control (negative), FLAG-TRIM71-WT, FLAG-TRIM71-R595H and DHX36 (positive control) showing protein-bound RNA. Boxes indicate expected size of TRIM71 protein or DHX36 protein. g–i, CLIP-qRT–PCR enrichment of CDKN1A or EGR1 upon co-precipitation with Flag-Ctrl and human Flag-TRIM71-WT, −ΔNHL6, −R608H and -R796H in HEK293T cells (g, h) or with endogenous mESC FLAG-TRIM71 and FLAG-TRIM71-R595H (I). Precipitated mRNA levels were normalized to input and to enrichment of housekeeping. j, qRT–PCR showing Cdkn1a and Egr1 levels of mutant mESCs relative to respective controls. k, Stepwise approach to identify novel RNA targets of TRIM71. Significance was calculated by two-sided Fisher’s exact test (GO analysis) or hypergeometric test (cell type enrichment analysis). Box plot (in k3): median (line) and 25th and 75th percentiles (box); whiskers extend up to 1.5 times the interquartile range from the top (bottom) of the box to the furthest datum within that distance, with outliers plotted as individual points. Analyzed datasets from ref. 12 and ref. 47. For detailed statistical information (k3), see Supplementary Table 13. l, qRT–PCR of the six predicted TRIM71 targets from k of mutant mESCs relative to respective controls. m, CLIP-qRT–PCR enrichment of Inhbb, Cbfa2t2 and Spred1 upon co-precipitation with endogenous FLAG-TRIM71-WT and FLAG-TRIM71-R595H. Precipitated mRNA levels were normalized to input and to enrichment of housekeeping. Statistical significance was tested by one-sample t-test (e, g–j, l, m): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and NS (not significant): P > 0.05. Data are represented as mean ± s.e.m., overlaid with individual data points. For detailed statistical information (e, g–j, l, m), see Supplementary Table 13. Source data are provided.

Article Snippet: CDKN1A (Hs00355782_m1), Cdkn1a (Mm04205640_g1), Inhbb (Mm03023992_m1), Gapdh (Mm99999915_g1), Trim71 (Mm01341471_m1). qRT–PCR Primer:. qHs_EGR1-fw: CCTGACCGCAGAGTCTTTTC. qHs_EGR1-rv: AGCGGCCAGTATAGGTGATG. qMm-Egr1-for: ACCACAGAGTCCTTTTCTGACA. qMm-Egr1-rev:TGAAAAGGGGTTCAGGCCAC. qMm-Spred1-for:AGAAGCTCAGACATAAGACCGT. qMm-Spred1-rev:ATGTCTAGGCCTTGCTGACT. qMm-Cbfa2t2-for: CTCAGGCAGAGGGTCGAAG. qMm-Cbfa2t2-rev:GGCACCCTTTTCTCACAACC. qMm-Tcf4-for:TAGGGAAAGCCCTAGCTTCG. qMm-Tcf4-rev:GCCTGCTGAGAGTGAAGGAG. qMm-Crebbp-for:CATTGTCAGGCTGGGAAAGC. qMm-Crebbp-rev:AAAGGGAGGCAAACAGGACA. qMm-Bcl11b-for:GAATAGATGCCGGGGCAATG. qMm-Bcl11b-rev:ATGGTAGCCTCCACATGGTC. q-18S-rRNA-fw: GTAACCCGTTGAACCCCATTC. q-18S-rRNA-rv: CCATCCAATCGGTAGTAGCGAC.

Techniques: RNA Binding Assay, Immunostaining, Western Blot, Ubiquitin Proteomics, Quantitation Assay, Immunoprecipitation, Autoradiography, Control, Positive Control, Quantitative RT-PCR, Mutagenesis