DOI: 10.5281/zenodo.14163382
title: Efficient globin production during terminal erythropoiesis depends on the synergistic action of TENT5C poly(A) polymerase and LARP4B

description: This repository contains supplementary data accompanying related publication.

 Our preprint can be found here: https://doi.org/10.1101/2024.11.14.623596
description: Red blood cell development is a unique process in which reduced transcriptome and proteome complexity enable extensive hemoglobin production. Here, we describe the cooperative roles of cytoplasmic poly(A) polymerase TENT5C and the poly(A) tail-protecting LARP4B RNA-binding protein in ensuring proper hemoglobin synthesis. TENT5C catalytic mutant knock-in mice exhibit microcytic hypochromic anemia similar to the constitutive knockout. TENT5C counteracts gradual deadenylation of globin mRNA during erythropoiesis. In the late stages, TENT5C dysfunction results in globin poly(A) tail shortening and a pronounced reduction in mRNA levels in reticulocytes. Proteomic experiments revealed a transient but specific association of TENT5C with LARP4B. LARP4B depletion leads to downregulation and poly(A) tail shortening of globin mRNAs. Furthermore, loss of TENT5C catalytic activity is accompanied by compensatory upregulation of LARP4B. Finally, precise regulation of globin poly(A) tails by deadenylation and readenylation is highlighted by the destabilization of TENT5C by the CCR4-NOT deadenylase complex-associated E3 ubiquitin ligase CNOT4.
description: Tables



Supplementary_table_1_Differential_adenylation_E14.5.xlsx - Excel file with differential adenylation results (WT vs Tent5c KO, E14.5 FLEB).

Supplementary_table_2_Differential_adenylation_BasoE_OrthoE_PolyE_Retc.xlsx - Excel file with differential adenylation results (each sheet contains one  comparison for BasoE, PolyE, OrthoE, Retc for WT vs Tent5c catalytic mutant, respectively).

Supplementary_table_3_TurboID_assay.xlsx - Excel file with TurboID analysis summaries.

Supplementary_table_4_Differential_adenylation_Tent5c_Larp4ab_silencing.xlsx - Excel file with differential adenylation results (control shRNA vs given silencing with either one or multiple shRNA, respectively).

Supplementary_table_5_Degronopedia_analysis.xlsx - Excel file with Degronopedia analysis overview.

Supplementary_table_6_Key_resources.xlsx - Key resource Excel file



Raw data



Supplementary_Resource_1.tar.gz - TurboID assay MS/MS raw data 

Supplementary_Resource_2.tar.gz - Nanopolish polya predictions for FLEB E14.5 data

Supplementary_Resource_3.tar.gz - Dorado polya predictions for sorted BasoE, OrthoE, PolyE and Retc data

Supplementary_Resource_4.tar.gz - Dorado polya predictions for silencing experiment data



Figures



Graphical abstract.pdf - a visual summary of the study

Fig.1.pdf - TENT5C catalytic activity is required for normal erythropoiesis.

Fig.2.pdf - TENT5C inactivity induces splenic stress erythropoiesis.

Fig.3.pdf - TENT5C counteracts globin mRNA degradation during late erythropoiesis

Fig.4.pdf - Globin poly(A) dynamics during erythropoiesis. 

Fig.5.pdf - LARP4B as a novel interactor determining stability of TENT5C

Fig.6.pdf - Functional association of LARP4A/4B with globin mRNAs stability regulation

Fig.7.pdf - CNOT4 drives TENT5C instability



Supplementary figures



Fig.S1.pdf - TENT5C-dependent anemic phenotype is not related to iron pathways

Fig.S2.pdf - HBA, HBB, PCBP1, PCBP2 and TENT5C expression ex vivo and evaluation of putative TENT5C substrates suggested by Yang et al. 

Fig.S3.pdf - Sequence alignment of mouse hemoglobin transcripts. Reference according to Gencode VM32, generated using MAFFT (v7.511)

Fig.S4.pdf - TurboID supplementary information

Fig.S5.pdf - Supplementary data on shRNA silencing

Fig.S6.pdf - Globin expression pattern during LARP4A, LARP4B and/or TENT5C shRNA silencing

Fig.S7.pdf - TENT5C degradation pathways and supplementary data on CNOT4 silencing

Fig.S8_gating ABC.pdf - Gating strategy used in experiments (part 1)

Fig.S8_gating DE.pdf - Gating strategy used in experiments (part 2)



Additional resources (raw data underlying figures etc.)



Cytometry_raw.tar - raw data from cytometry experiments

ELISA.tar - raw data from ELISA experiments

Procyte_Dx.tar - raw data from blood parameters assessment

RT_qPCR.tar - raw dara from RT-qPCR experiments

WB.tar - raw data from western blotting




 
description: Detailed description of provided files:



Supplementary_table_1_Differential_adenylation_E14.5.xlsx - Excel file with differential adenylation results (WT vs Tent5c KO, E14.5 FLEB). Statistical significance was assessed using the two-sided Wilcoxon signed-rank test (α = 0.05). Table contains the following columns:



ensembl_transcript - transcript identifier in Ensembl format  

p.value - statistical significance calculated using a two-sided Wilcoxon rank-sum test with alpha = 0.05 

stats_code - a quality indicator showing whether read coverage in both conditions was adequate to support reliable statistical inference  

cohen_d - effect size (an auxiliary metric that helps discern transcripts with differences in poly-A tail length between conditions, even when statistical significance may be driven primarily by high read counts) 

*_counts - number of mapped reads in WT and mutant samples, respectively

*_polya_gm_mean - geometric mean of poly(A) tail length in each condition, respectively

length_diff - a measure of the change in poly(A) tail length between WT and mutant samples [nt]

fold_change - the magnitude of length_diff

padj - adjusted p-value (FDR-corrected) controlling for multiple testing significance

effect_size - descriptive measure of the magnitude of the change

significance - categorical label (e.g., FDR<0.05 / NotSig) based on padj threshold indicating whether the observed difference is statistically significant

ensembl_gene_id - gene identifier corresponding to the transcript in Ensembl format  

transcript_biotype - classification of transcript type (protein_coding, lncRNA, pseudogene, etc.).

description - short functional summary of each gene 

gene_name - gene symbol



Supplementary_table_2_Differential_adenylation_BasoE_OrthoE_PolyE_Retc.xlsx - Excel file with differential adenylation results (each sheet contains one  comparison for BasoE, PolyE, OrthoE, Retc for WT vs Tent5c catalytic mutant, respectively). Statistical significance was assessed using the two-sided Wilcoxon signed-rank test (α = 0.05). Each of the subtable (sheet) contains the following columns:



ensembl_transcript - transcript identifier in Ensembl format  

p.value - statistical significance calculated using a two-sided Wilcoxon rank-sum test with alpha = 0.05 

stats_code - a quality indicator showing whether read coverage in both conditions was adequate to support reliable statistical inference  

cohen_d - effect size (an auxiliary metric that helps discern transcripts with differences in poly-A tail length between conditions, even when statistical significance may be driven primarily by high read counts) 

*_counts - number of mapped reads in WT and mutant samples, respectively

*_polya_gm_mean - geometric mean of poly(A) tail length in each condition, respectively

length_diff - a measure of the change in poly(A) tail length between WT and mutant samples [nt]

fold_change - the magnitude of length_diff

padj - adjusted p-value (FDR-corrected) controlling for multiple testing significance

effect_size - descriptive measure of the magnitude of the change

significance - categorical label (e.g., FDR<0.05 / NotSig) based on padj threshold indicating whether the observed difference is statistically significant

ensembl_gene_id - gene identifier corresponding to the transcript in Ensembl format  

transcript_biotype - classification of transcript type (protein_coding, lncRNA, pseudogene, etc.).

description - short functional summary of each gene 

gene_name - gene symbol



Supplementary_table_3_TurboID_assay.xlsx - Excel file with TurboID analysis summaries. Contains the following sheets:



all proteins - all identified proteins in the TurboID assay. The table reports results for each identified protein, including basic annotation (gene name, protein ID, description, organism, sequence length, and coverage), quality and identification metrics (protein existence, probability, top peptide probability, number of peptides), and detailed quantitative measurements across replicates. Quantitative data include spectral counts, unique spectral counts, total intensities, unique intensities, MaxLFQ intensities, and relative intensities for both Tent5c and WT samples, enabling comparison of protein enrichment and reproducibility across biological replicates.

replicates overlap - overlap between 3 replicates. Table summarizes the quantitative and qualitative data from the TurboID proteomics experiment for each identified protein. It includes protein annotation (gene, protein ID, sequence length, coverage, organism, description), identification confidence metrics (protein existence, probabilities, peptide counts), and detailed quantitative measurements across all biological replicates. Quantitative data encompass spectral counts, unique counts, total and unique intensities, MaxLFQ intensities, and relative abundance and specificity metrics for Tent5c and WT samples.

GO terms - statistically significant GO terms for overlapping proteins with average specificity > 0. Contains following columns:



significant – indicates whether the GO term is statistically significant (TRUE/FALSE)

p_value – raw p-value for the enrichment of the term

term_size – total number of genes associated with the GO term in the reference set

query_size – number of genes in the query set tested for enrichment

intersection_size – number of genes shared between the query set and the GO term

precision – proportion of query genes in the intersection relative to the query set (intersection_size/query_size)

recall – proportion of term genes captured by the intersection (intersection_size/term_size)

term_id – GO identifier for the term (e.g., GO:0043604)

source – ontology source (e.g., GO:BP for Biological Process)

term_name – descriptive name of the GO term

effective_domain_size – total number of genes in the background/reference used for enrichment calculation

source_order – internal ordering or index of the GO term within the source database



differential abundance - proteins with estimated changes and associated statistics. Contains the following columns:



gene – gene symbol of the protein

protein_id – database identifier 

comparison – experimental contrast tested 

missingness – type of missing data: MNAR, MAR, or complete

diff – estimated difference in abundance between groups

CI_2.5 – lower bound of 95% confidence interval for diff

CI_97.5 – upper bound of 95% confidence interval for diff

avg_abundance – average abundance across all samples

t_statistic – t-statistic for the comparison

pval – raw p-value

adj_pval – multiple testing–corrected p-value

B – log-odds of differential abundance 

n_obs – number of observations used per protein





Supplementary_table_4_Differential_adenylation_Tent5c_Larp4ab_silencing.xlsx - Excel file with differential adenylation results (each sheet contains one  comparison for control shRNA vs given silencing with either one or multiple shRNA, respectively). Statistical significance was assessed using the two-sided Wilcoxon signed-rank test (α = 0.05). Each of the subtable (sheet) contains the following columns:



ensembl_transcript - transcript identifier in Ensembl format  

p.value - statistical significance calculated using a two-sided Wilcoxon rank-sum test with alpha = 0.05 

stats_code - a quality indicator showing whether read coverage in both conditions was adequate to support reliable statistical inference  

cohen_d - effect size (an auxiliary metric that helps discern transcripts with differences in poly-A tail length between conditions, even when statistical significance may be driven primarily by high read counts) 

*_counts - number of mapped reads in control and silenced samples, respectively

*_polya_gm_mean - geometric mean of poly(A) tail length in each condition, respectively

length_diff - a measure of the change in poly(A) tail length between control and silenced samples [nt]

fold_change - the magnitude of length_diff

padj - adjusted p-value (FDR-corrected) controlling for multiple testing significance

effect_size - descriptive measure of the magnitude of the change

significance - categorical label (e.g., FDR<0.05 / NotSig) based on padj threshold indicating whether the observed difference is statistically significant

ensembl_gene_id - gene identifier corresponding to the transcript in Ensembl format  

transcript_biotype - classification of transcript type (protein_coding, lncRNA, pseudogene, etc.).

description - short functional summary of each gene 

gene_name - gene symbol



Supplementary_table_5_Degronopedia_analysis.xlsx - Excel file with Degronopedia analysis overview:



degronopedia_overview - Overview of used input and method details used in degronopedia analysis

degron_data - Identified degron motifs

degron_conservation - Degron conservation

consurf_evo_conservation - Evolutionary conservation of TENT5C aa sequence scored by ConSurf (The table shows the residue variety in % for each position in the query sequence, each column shows the % for that amino acid found in position in the MSA).



Supplementary_table_6_Key_resources.xlsx - Key resource Excel file with 2 sheets:



Resources - table listing resources and reagents used in this study:



antibodies,

bacterial and viral strains

chemicals, peptides & proteins

critical commercial assays

experimental model cell lines

experimental model organisms

oligonucleotides

recombinant DNA

software and algorithms.



Sequenced_samples - metadata of all samples sequenced in this study, containing following informations: 



experiment - the experimental context for which the sample was sequenced

sample_alias - the revised sample identifier following review (e.g., Larp4 updated to Larp4a; Larp5 updated to Larp4b)

sample_title - sample ID in European Nucleotide Archive (ENA)

sequencing_type - sequencing protocol (either cDNA or DRS)

kit - sequencing chemistry used to prepare the library

reads - count of reads produced

Guppy - version of Gupy basecaller used

Nanopolish - version of Nanopolish polya used

Dorado - version of Dorado basecaller used

reference - version of reference sequence used

organism - organism the sample was derived from

accession - sample accession number in European Nucleotide Archive (ENA)

project - project accession number in European Nucleotide Archive (ENA)

sample_description - a description of the sample contents and the preparation methodology





Supplementary_Resource_1.tar.gz - TurboID assay MS/MS raw data, each file in the archive corresponds to a biological replicate of either WT (control) or Tent5c-EGFP.

Supplementary_Resource_2.tar.gz - Nanopolish polya predictions for FLEB E14.5 data (corresponding to Supplementary table 1). Contains .tsv files one per each sequenced sample, with naming convention corresponding to sample aliases in Supplementary table 6. Each of the .tsv file within the archive contains the following columns:



readname - unique read ID from the fast5 file

contig - contig/sequence in the reference that the read aligns to

position - position on the reference contig at which the read-alignment starts

leader_start - index of the raw (pico-amp) sample at which segmentation algorithm declares the leader starts

adapter_start - index of the raw (pico-amp) sample at which segmentation algorithm declares the sequencing adapter region starts

polya_start - index of the raw (pico-amp) sample at which segmentation algorithm declares the poly(A) region of the RNA starts

transcript_start - index of the raw (pico-amp) sample at which segmentation algorithm declares the transcript body region of the RNA starts

read_rate - number of nucleotides moving through the pore per second

polya_length - estimated poly(A) tail length [nt]

qc_tag - quality tag assigned by nanopolish polya function



Supplementary_Resource_3.tar.gz - Dorado polya predictions for sorted BasoE, OrthoE, PolyE and Retc data (corresponding to Supplementary table 2). Contains .tsv files one per each sequenced sample, with naming convention corresponding to sample aliases in Supplementary table 6. Each of the .tsv file within the archive contains the following columns:



read_id - unique read ID from the pod5 file

reference - contig/sequence in the reference that the read aligns to

ref_start - position on the reference contig at which the read-alignment starts

ref_end - position on the reference contig at which the read-alignment ends

mapq - alignment mapping quality

pt - estimated poly(A) tail length [nt]

sequence - basecalled sequence of read



Supplementary_Resource_4.tar.gz - Dorado polya predictions for silencing experiment data (corresponding to Supplementary table 4). Contains .tsv files one per each sequenced sample, with naming convention corresponding to sample aliases in Supplementary table 6. Each of the .tsv file within the archive contains the following columns:



read_id - unique read ID from the pod5 file

reference - contig/sequence in the reference that the read aligns to

ref_start - position on the reference contig at which the read-alignment starts

ref_end - position on the reference contig at which the read-alignment ends

mapq - alignment mapping quality

pt - estimated poly(A) tail length [nt]

sequence - basecalled sequence of read



Cytometry_raw.tar - raw data from cytometry experiments



archive containing subfolders corresponding to each experiment. Within each subfolder, fcs files are stored.



ELISA.tar - raw data from ELISA experiments



xlsx files with the reports from Assayfit Pro 1.41 and Magellan measurements



Procyte_Dx.tar - raw data from blood parameters assessment



Results from ProCyte Dx hematology analysis of WT vs TENT5Ccat mice in xlsx format



RT_qPCR.tar - raw dara from RT-qPCR experiments



raw and merged results in xlsx format 



WB.tar - raw data from western blotting