riboviz 2:A flexible and robust ribosome profiling data analysis and visualization workflow

MOTIVATION: Ribosome profiling, or Ribo-seq, is the state-of-the-art method for quantifying protein synthesis in living cells. Computational analysis of Ribo-seq data remains challenging due to the complexity of the procedure, as well as variations introduced for specific organisms or specialized analyses. RESULTS: We present riboviz 2, an updated riboviz package, for the comprehensive transcript-centric analysis and visualization of Ribo-seq data. riboviz 2 includes an analysis workflow built on the Nextflow workflow management system for end-to-end processing of Ribo-seq data. riboviz 2 has been extensively tested on diverse species and library preparation strategies, including multiplexed samples. riboviz 2 is flexible and uses open, documented file formats, allowing users to integrate new analyses with the pipeline. AVAILABILITY AND IMPLEMENTATION: riboviz 2 is freely available at github.com/riboviz/riboviz

Celf4 controls mRNA translation underlying synaptic development in the prenatal mammalian neocortex

Abstract Abnormalities in neocortical and synaptic development are linked to neurodevelopmental disorders. However, the molecular and cellular mechanisms governing initial synapse formation in the prenatal neocortex remain poorly understood. Using polysome profiling coupled with snRNAseq on human cortical samples at various fetal phases, we identify human mRNAs, including those encoding synaptic proteins, with finely controlled translation in distinct cell populations of developing frontal neocortices. Examination of murine and human neocortex reveals that the RNA binding protein and translational regulator, CELF4, is expressed in compartments enriched in initial synaptogenesis: the marginal zone and the subplate. We also find that Celf4/CELF4-target mRNAs are encoded by risk genes for adverse neurodevelopmental outcomes translating into synaptic proteins. Surprisingly, deleting Celf4 in the forebrain disrupts the balance of subplate synapses in a sex-specific fashion. This highlights the significance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, potentially contributing to sex differences

Supplemental Figure S2. Extracellular vesicles (EVs) from SW480 IMP1Null cells isolated by size-exclusion chromatography (SEC).

Supplemental Figure 2. Extracellular vesicles (EVs) from SW480 IMP1Null cells isolated by size-exclusion chromatography (SEC). Fractions (fx) 3-15 are shown. For Fx 3-11, 70µL of sample was concentrated by boiling and loaded on the gel. Protein concentration was below detection for these samples. For Fx 12-15, 15µg of protein were loaded. HT-29 IMP1OE whole cell lysate (WCL) was used as the loading control. Western blotting was used to detect tetraspanin protein and EV marker, CD9 and determine which SEC fractions to collect for subsequent experiments. </p

Supplemental Figure S3. Densitometry from whole cell lysate (WCL) and extracellular vesicle (EV) western blots.

Supplemental Figure S3. Densitometry from whole cell lysate (WCL) and extracellular vesicle (EV) western blots. A) Western blots for accessory protein ALIX, tetraspanin CD63, and secretion mediator Rab27A were performed on WCL isolated from HT-29 and SW480 null and IMP1OE cell lines. Densitometry was performed using the area under the curve function with ImageJ v.2.1.0. Data are presented as the mean and SEM from n=3 independent passages of each cell line and compared using unpaired student’s t-test with *p<0.05 being considered statistically significant. B) Western blots for common EV cargo proteins (CD63, TSG101, CD9) and Golgi protein GM130 in EV lysates isolated from conditioned media from HT-29 and SW480 null and IMP1OE cell lines. Densitometry was performed using the image analysis function on LI-COR Image Studio Lite v.5.2.5. Data are presented as the mean and SEM from n=3 independent passages of HT-29 cell lines and n=2 passages of SW480 cells lines and compared using  unpaired student’s t-test with *p<0.05 being considered statistically significant.</p