Analysis of Quick irCLIP of eIF3 to RNAs in undifferentiated and differentiated neural progenitor cells.

A) Structure of the human eIF3 complex as part of the 48S initiation complex. PDB: 6ZMW (Brito Querido et al., 2020). B) Generation of NPCs from hPSCs by embryoid body (EB) and neural rosette selections. C) Western blots of neural markers Pax6 and Sox1 and pluripotent marker Oct4 from hPSCs and hPSC-derived NPCs (NPCs corresponding to passages 1, 2 and 7 are shown). D) Western blots of puromycin-treated (15 min) NPCs. Total protein stain is shown as loading control. Differentiated NPCs were treated with forebrain neuron differentiation medium for the indicated time points. Undifferentiated NPCs were treated with NPC medium for the indicated time points. The ratios of puromycin signal to total protein levels can be found in Figure 1–figure supplement 1A. E) Schematic of Quick-irCLIP. F) Schematic of the NPC treatment performed in the Quick-irCLIP, APA-Seq, Ribosome profiling, and mRNA-Seq experiments. Treatment consisted of change of media and incubation at 37 °C for 2 hrs. Differentiation media was used for differentiated (Diff.) NPCs and Basal media for undifferentiated (Undiff.) NPCs. G) Immunoprecipitation samples of assembled eIF3 complexes from undifferentiated NPCs. 5% inputs (In), flowthroughs (Ft), and eluates (Elu) are shown. Western blot for subunit EIF3G is not shown because we could not identify an effective antibody for its detection. EIF3J, not shown, is usually dissociated from the eIF3 complex upon immunoprecipitation. However, we detected EIF3G by mass spectrometry in the IPs (Supplementary file 1). H) Infrared (IR) image of IR dye-labeled, eIF3 UV-crosslinked RNA transcripts from Diff. and Undiff. NPCs. Regions marked with red boxes, which correspond to subunits EIF3A through EIF3D, were excised from the blot. I) Biological Function enrichment determined using the STRING database for the undifferentiated NPC biological replicates of the EIF3A/B/C/D Quick-irCLIP libraries, using the top 500 mRNA hits from the crosslinking analysis. J) Categories of RNAs crosslinked to eIF3 in undifferentiated and differentiated NPCs, for 3 replicates plotted in log-scale with standard deviation error bars. K) mRNA regions that crosslink to eIF3 in undifferentiated and differentiated NPCs. L) Crosslinking of eIF3 across the transcripts of NES and VIM mRNAs in differentiated and undifferentiated NPCs. Read coverage is provided in Counts Per Million (CPM).

NPCs treated with differentiation media or kept in undifferentiated media (2-hour treatment).

A) Levels of protein synthesis in undifferentiated and differentiated NPCs at different time points, as quantified by puromycin incorporation. Puromycin signal was normalized to total protein levels, which were measured with total protein stain. This experiment is representative of multiple timecourses. B) Cellular morphology of the NPCs using dark field microscopy.

Venn Diagram of genes identified by Quick-irCLIP (differentiated NPCs) and PAR-CLIP (HEK293T and activated Jurkat T cells).

The top ∼ 200 mRNA transcripts from the three libraries ranked by total reads mapped to a given gene are compared (See Materials and Methods).

Crosslinking of eIF3 in undifferentiated NPCs and in HEK293T cells.

Crosslinks in undifferentiated NPCs are in blue, and in HEK294T cells in red. Shown are the 5’-UTR regions of A) CCND2 and B) TUBB mRNAs.

Crosslinking of eIF3 in differentiated and undifferentiated NPCs across the 3’-UTR regions of ACTG1 and FTL mRNAs.

APA-Seq data is also shown for both transcripts.

Titration of RNAse I for the Quick-irCLIP experiment.

Crosslinked samples were treated with RNAse I for 3 minutes and subject to anti-EIF3B IP. Shown are 5% input of the lysate, flowthrough (FTH) and elution (ELU) for each concentration of RNAse I. Molecular weight markers (MW, M) are shown to the left.

Correlation of Quick-irCLIP replicates.

Spearman correlation coefficients (Rho) of irCLIP bioreplicates (n=3) for differentiated (average Rho: 0.7855, standard deviation: 0.0092) and undifferentiated NPCs (average Rho: 0.834, standard deviation: 0.0058) demonstrate high reproducibility.

Crosslinking of eIF3 to mRNA 3’-UTRs adjacent to the poly(A) tail.

A) Crosslinking of eIF3 across the TUBB mRNA in undifferentiated NPCs. A zoomed-in view of TUBB mRNA 3’-UTR terminus with the eIF3 crosslinks is shown. The polyadenylation signal (PAS) is marked. B) Crosslinking of eIF3 across the APP mRNA 3’-UTR in undifferentiated NPCs. A zoomed-in view of the two 3’-UTR regions presenting eIF3 crosslinks are shown as well as their PAS sequences. C) Sequence logo of the eIF3 crosslinks located at 3’-UTRs in differentiated and undifferentiated NPCs. D) Crosslinking of eIF3, APA-Seq peaks, and mRNA-Seq across the MAP1B, S100B, TUBB and APP mRNAs in differentiated and undifferentiated NPCs.

Motif enrichment of eIF3 3’-UTR crosslinks for undifferentiated and differentiated NPCs.

Motifs were identified with HOMER (v4.11).

Examples of eIF3 crosslinking to 3’-UTR regions of mRNAs.

A) Crosslinking of eIF3 in undifferentiated NPCs as indicated across the 3’-UTR region of NES mRNA. APA-Seq data is also shown. B) Crosslinking of eIF3 in undifferentiated NPCs as indicated across the 3’-UTR region of VIM mRNA. APA-Seq data is also shown.

Ribosome profiling of undifferentiated and differentiated NPCs, and comparisons to eIF3 Quick irCLIP crosslinking.

A) Crosslinking of eIF3 across the ID2 mRNA in undifferentiated and differentiated NPCs. APA-Seq and mRNA-Seq data are also shown. B) log2 fold change of ribosomal footprints and log2 fold change of mRNA transcript levels upon NPC differentiation. Number of samples within each condition is shown. C) Ribosomal footprints across the ID2 mRNA in undifferentiated and differentiated NPCs. mRNA-Seq data is also shown. D) Western blots of ID2, SLC38A2 (SNAT2) and Hsp90 in undifferentiated and differentiated NPCs. E) Crosslinking of eIF3 to the 3’-UTR of the EEF1A and ACTB mRNAs in undifferentiated NPCs. APA-Seq data is also shown. F) Cumulative distribution of the log2 fold change in ribosome occupancy after differentiated cells for genes found to have either an increase (blue) or decrease (red) in 3’-UTR irCLIP coverage upon differentiation, excluding genes with 3’-UTR less than 50 nts and without sufficient coverage to compute an adjusted p-value in both datasets. ECDF: empirical cumulative distribution function.

TBE 8% Urea-PAGE of the OTTR reaction products of cDNA libraries constructed from undifferentiated and differentiated NPCs.

OTTR was also performed with control samples that yield marker products of 30, 40, 60, 80 nts in size (marked in yellow) so they could be used as references for gel excision for monosome and disome libraries. Gel excisions performed are shown in red (monosome) and blue (disome) rectangles.

Ribosome profiling of undifferentiated and differentiated NPCs.

A) Fraction of sequencing reads mapped to each transcript type from monosome and disome profiling from P1 nuclease digested NPCs (undifferentiated and differentiated) and OTTR library cDNA synthesis. B) Framing among monosome libraries in undifferentiated and differentiated NPCs. C) Readlength of footprints aligned to CDS or terminating codons among disome libraries for undifferentiated and differentiated NPCs. D) A-site corrected initiation and termination profiles for genes with sufficient coverage (≥1 read per codon) for monosome and disome libraries. E) DESeq2 log2 fold change in CDS occupancy for disome libraries with respect to monosome libraries, stratified by CDS length in nucleotides.

Western blots of SLC38A2 for additional replicates of differentiated and undifferentiated NPCs.

Correlation of Quick-irCLIP and Translational Efficiency.

The Spearman correlation of average irCLIP 3’-UTR RPKM and mean TE across replicates (n=3) for transcripts with high and low differential TE ( p-value < 0.05) in differentiated and undifferentiated NPCs.

Crosslinking of eIF3 to polyadenylated mRNAs and model for mRNA circularization.

A) Crosslinking of eIF3 across the canonical non-polyadenylated histone H2AC11 mRNA and the variant polyadenylated histone H3-3B mRNA in undifferentiated NPCs. APA-Seq and mRNA-Seq data for these regions are also shown. B) Western blots of EIF3B immunoprecipitations from undifferentiated NPCs. C) Model for eIF3 contribution to mRNA circularization in highly translated transcripts.

Mechanism of eIF3 binding to mRNA 3’-UTR elements.

A) Crosslinking of eIF3 to histone mRNAs iin undifferentiated NPCs. Shown are canonical non-polyadenylated histone H3C1 and H4-16 mRNAs and the variant polyadenylated histone H2AZ1 mRNA. B) Western blots for EIF3B immunoprecipitation samples from UV-crosslinked HEK293T cells. C) Western blots for EIF3B immunoprecipitation samples from DSP-treated undifferentiated NPCs and HEK293T.

Crosslinking of eIF3 to mRNA 3’-UTRs of various lengths in NPCs.

Crosslinking patterns of eIF3 in differentiated and undifferentiated NPCs across RPS10, GAPDH, ACTG1, and TUBB mRNAs are shown. The length of each 3’-UTR is indicated. APA-Seq and mRNA-Seq data are also shown.