Detailed description of provided files: Supplementary_Data_1.xlsx- Excel file containing the numerical data underlying the figures and supplementary figures. Each tab (worksheet) contains the data corresponding to the figure or panel indicated by the worksheet name. Column names vary depending on the content of the respective plots. Supplementary_Data_2.xlsx - Excel file with differential adenylation results (each sheet contains one comparison for WT vs Tent5c KO, E14.5 FLEB, BasoE, PolyE, OrthoE, Retc for WT vs Tent5c catalytic mutant, respectively, 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). 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. 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_Data_4.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_Data_5.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_Data_6.zip - raw data from ELISA experiments xlsx files with the reports from Assayfit Pro 1.41 and Magellan measurements Supplementary_Data_7.zip - raw data from cytometry experiments archive containing subfolders corresponding to each experiment. Within each subfolder, fcs files are stored. Supplementary_Data_8.zip - raw data from blood parameters assessment Results from ProCyte Dx hematology analysis of WT vs TENT5Ccat mice in xlsx format Supplementary_Data_9.gz - Nanopolish polya predictions for FLEB E14.5 data corresponding to Supplementary_Data_2.xlsx. Contains .tsv files one per each sequenced sample, with naming convention corresponding to sample aliases in Supplementary_Data_5.xlsx. 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_Data_10.gz - Dorado polya predictions for sorted BasoE, OrthoE, PolyE and Retc data (corresponding to Supplementary_Data_2.xlsx). Contains .tsv files one per each sequenced sample, with naming convention corresponding to sample aliases in Supplementary_Data_5.xlsx. 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_Data_11.gz - Dorado polya predictions for silencing experiment data (corresponding to Supplementary_Data_2.xlsx). Contains .tsv files one per each sequenced sample, with naming convention corresponding to sample aliases in Supplementary_Data_5.xlsx. 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_Data_12.zip - raw dara from RT-qPCR experiments raw and merged results in xlsx format Supplementary_Data_13.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_Information.pdf - Supplementary figures merged with uncropped western blot images. 

Tables Supplementary_Data_1.xlsx - Data underlying plots/figures Supplementary_Data_2.xlsx - Excel file with differential adenylation results (each sheet contains one comparison for WT vs Tent5c KO, E14.5 FLEB, BasoE, PolyE, OrthoE, Retc for WT vs Tent5c catalytic mutant, control shRNA vs given silencing with either one or multiple shRNA, respectively) Supplementary_Data_3.xlsx - Excel file with TurboID analysis summaries Supplementary_Data_4.xlsx - Excel file with Degronopedia analysis overview Supplementary_Data_5.xlsx - Key resource Excel file Raw data Supplementary_Data_6.zip - raw data from ELISA experiments Supplementary_Data_7.zip - raw data from cytometry experiments Supplementary_Data_8.zip - raw data from blood parameters assessment Supplementary_Data_9.gz - Nanopolish polya predictions for FLEB E14.5 data Supplementary_Data_10.gz - Dorado polya predictions for sorted BasoE, OrthoE, PolyE and Retc data Supplementary_Data_11.gz - Dorado polya predictions for silencing experiment data Supplementary_Data_12.zip - raw data from RT-qPCR experiments Supplementary_Data_13.gz - TurboID assay MS/MS raw data Figures Figure_1.pdf - TENT5C catalytic activity is required for normal erythropoiesis Figure_2.pdf - TENT5C inactivity induces splenic stress erythropoiesis Figure_3.pdf - TENT5C counteracts globin mRNA degradation during late erythropoiesis Figure_4.pdf - Globin poly(A) dynamics during erythropoiesis Figure_5.pdf - LARP4B as an interactor determining stability of TENT5C Figure_6.pdf - Functional association of LARP4A/4B with globin mRNAs stability regulation Figure_7.pdf - CNOT4 drives TENT5C instability Supplementary figures Supplementary_Figure_1.pdf - TENT5C-dependent anemic phenotype is not related to iron pathways Supplementary_Figure_2.pdf - HBA, HBB, PCBP1, PCBP2 and TENT5C expression ex vivo and evaluation of putative TENT5C substrates suggested by Yang et al. Supplementary_Figure_3.pdf - TurboID supplementary information Supplementary_Figure_4.pdf - Validation of LARP4A, LARP4B and TENT5C knockdown Supplementary_Figure_5.pdf - Globin poly(A) tail length distribution pattern upon LARP4A, LARP4B and/or TENT5C silencing Supplementary_Figure_6.pdf - Transcript-level effects of LARP4A/LARP4B and TENT5C perturbation Supplementary_Figure_7.pdf - TENT5C degradation pathways and CNOT4 silencing Supplementary_Figure_8_gating ABC.pdf - Gating strategies Supplementary_Figure_8_gating DE.pdf - Gating strategies Supplementary_Figure_9.pdf - Sequence alignment of mouse hemoglobin transcripts Additional resources Graphical_abstract.pdf - a visual summary of the study Supplementary_Information.pdf - Supplementary figures merged with uncropped western blots

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. Through poly(A) tail extension, TENT5C counteracts the 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. Consistent with this interaction, LARP4B depletion resulted in reduced globin mRNA abundance and shortened poly(A) tails, which proves a novel physiological role for this RNA binding protein. Furthermore, we show that TENT5C is a highly unstable protein whose stability is partially dependent on CNOT4, a deadenylase-associated E3 ubiquitin ligase.

This repository contains supplementary data accompanying related publication. Our preprint can be found here: https://doi.org/10.1101/2024.11.14.623596