Affinity purification of ribosomal complexes from purified somata of SCPN and CPN in vivo

(A) Workflow to investigate ribosomal complexes from specific cortical projection neuron (PN) subtypes in vivo.

(B) Dual retrograde labeling of CPN and SCPN at P1 in the same mice distinctly labels the appropriate, non-overlapping subpopulations of cortical PN by P3. Inset B’ shows the location of cortical layers II/III and V, striatum, and corpus callosum (CC). (n=4 biological replicates)

(C) Bioanalyzer characterization shows specific recovery of rRNAs from both FACS-purified CPN and SCPN using rRNA IP.

(D) Quantification of 28S and 18S rRNA recovery following IP with rRNA antibody or isotype control antibody in purified CPN and SCPN. Individual dots represent biological replicates, with bars showing mean values and error bars indicating standard deviation.

(E) Mass spectrometry analysis shows enrichment of most ribosomal proteins (76-77 out of ∼80 known proteins), translation factors, and known ribosome-associated proteins (RAPs) in rRNA IP samples compared to control IP samples from both SCPN and CPN. RAPs were reported in Simsek et al. 201725 for mESC, Bartsch et al. 202330 for hESC, and Susanto et al. 202431 for E12.5 mouse forebrain.

(F) PANTHER protein classification analyses of proteins found in at least 3 rRNA IP replicates but absent in control IP samples, excluding core ribosomal proteins, translation factors, and known RAPs, reveal significant overrepresentation of proteins involved in RNA metabolism and translation for both subtypes.

SCPN and CPN ribosomal complexes share similar RP composition, but interact with distinct associated proteins

(A) Label-free quantification proteomic analysis comparing SCPN and CPN ribosomal complexes: All identified RPs have detectable abundance in ribosomes of both subtypes. 16 associated proteins are detected exclusively in 3 or more replicates from SCPN.

(B) List of 16 proteins detected exclusively in 3 or more replicates of rRNA IP from SCPN

(C) Examples of extracted ion chromatograms of peptides from proteins that are detected exclusively in SCPN rRNA IP, showing only samples with detected abundance of indicated peptides. Peptides are referred to by their parent proteins and their amino acid sequence locations within their protein. Vertical lines indicate retention times of MS2 spectra used for peptide identification, with colors corresponding to sample origins of the MS2 spectra. A set of proteins with distinct known functions is shown.

(D) Quantitative differential analysis of 478 proteins shared by at least 3 replicates of both subtypes: SCPN and CPN ribosomes share largely the same RP composition; 3 proteins enriched in CPN ribosomal complexes (adjusted p-value <=0.1)

Ribosome interaction for proteins identified exclusively in SCPN by rRNA pulldown, with validation of PRKCE-ribosome interaction

(A) Protein interaction network showing reported physical interactions of SCPN-specific RAPs with ribosomal components and with each other. Line colors and thicknesses indicate distinct methodologies and number of interactions with ribosomal proteins (RPs) identified, respectively. Data were retrieved from BioGrid40, OpenCell41, Simsek et al. 201725, and Bartsch et al. 202330.

(B) Co-immunoprecipitation of PRKCE and RPL22 (n = 3, biological replicates)

(C) Specific recovery of ribosomal RNA in PRKCE IP versus isotype control IP, shown by representative Bioanalyzer electropherograms.

(D) Quantification of 28S and 18S rRNA recovery following PRKCE IP versus paired isotype control IP.

Several proteins both identified exclusively in RCs of SCPN and with validated ribosome interaction exhibit enriched expression and concordant translation by SCPN

(A) Comparative transcriptomic analysis of purified SCPN and CPN somata, highlighting differentially enriched transcripts encoding validated SCPN-specific RAPs. Dataset from Froberg et al., 202321

(B) Comparative ribosome profiling of purified SCPN and CPN somata, highlighting transcripts encoding validated SCPN-specific RAPs with differential ribosome occupancy. Dataset from Froberg et al., 202321

(C) S100a10 ISH signal is limited to cortical layer V, the predominant laminar position of SCPN. Reproduced from Arlotta*, Molyneaux* et al. 2005 2, also reporting SCPN-specific expression compared with CPN.

© 2005, Elsevier. Figure 4C is reprinted from Figure 3K from Arlotta et al., 2005, with permission from Elsevier. It is not covered by the CC-BY 4.0 licence and further reproduction of this panel would need permission from the copyright holder.

rRNA IP approach recovers more rRNAs from FACS-purified CPN than an approach using RiboTag mice and HA IP

(A) Schematic of pulldown of ribosomal complexes using HA IP from CPN expressing Cre-induced HA-tagged RPL22 (RiboTag + HA IP approach), compared to rRNA IP approach. (B) rRNA IP recovers ∼3-fold more 28S and 18S rRNA than HA IP from RiboTag mice, using the same number of FACS-purified CPN, while maintaining low non-specific binding. Individual dots represent biological replicates, with bars showing mean values and error bars indicating standard deviation.

Bioinformatic normalization enables differential analysis between CPN and SCPN rRNA IPs replicate samples

Median-of-ratios normalization was applied to protein abundances to account for differences in protein input between replicate (Rep) samples. For missing values in 1-2 samples within each subtype group, CPN and SCPN samples were analyzed separately using k-nearest neighbor (kNN) imputation (k=2). Protein abundances were then log2-transformed.