Fig. 5

Nudt21 Directs PolyA Site Selection and Represses the mRNA Stability of Autophagy-Related Genes. A Histogram summarizing the number of genes associated with changes in the percent distal polyA site usage index (PDUI) in WT and Nudt21-cKO BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for the indicated durations. B Venn diagram showing the overlap of genes with changes in both 3’UTR length and mRNA expression levels in Nudt21-cKO BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for 4 hours. C Histogram of Gene Ontology (GO) enrichment analysis identifying autophagy-related pathways among the 354 genes with changes in PDUI and mRNA levels in Nudt21-cKO BMDMs compared with WT controls. D List of autophagy-related genes from the overlapping set shown in B. E RNA immunoprecipitation (RIP)-qPCR analysis showing the binding of Nudt21 to the mRNAs of Map1lc3b and Ulk2 in WT BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for 4 hours. F RNA-seq reads across the Map1lc3b and Ulk2 loci in WT (blue) and Nudt21-cKO (red) BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for 4 hours. G, H RT‒qPCR analysis of distal polyA site usage in the 3’UTRs of Map1lc3b (G) and Ulk2 (H) in WT and Nudt21-cKO BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for the indicated durations. I, J RT‒qPCR analysis of Map1lc3b (J) and Ulk2 (K) mRNA decay in WT and Nudt21-cKO BMDMs at multiple time points (1 hour and 2 hours) after 5 μM actinomycin D (ActD) treatment (n = 3 per group). The error bars represent the standard errors of the means (SEMs). P values were determined by unpaired Student’s t-test