Inhibitor AN3661 reveals biological functions of Arabidopsis CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 73.

Cleavage and polyadenylation specificity factor (CPSF) is a protein complex that plays an essential biochemical role in mRNA 3'-end formation, including poly(A) signal recognition and cleavage at the poly(A) site. However, its biological functions at the organismal level are mostly unknown in multicellular eukaryotes. Study of plant CPSF73 has been hampered by the lethality of Arabidopsis (Arabidopsis thaliana) homozygous mutants of AtCPSF73-I and AtCPSF73-II. Here, we used poly(A) tag sequencing (PAT-seq) to investigate the roles of AtCPSF73-I and AtCPSF73-II in Arabidopsis treated with AN3661, an antimalarial drug with specificity for parasite CPSF73 that is homologous to plant CPSF73. Direct seed germination on AN3661-treated medium was lethal; however, 7-day-old seedlings treated with AN3661 survived. AN3661 targeted AtCPSF73-I and AtCPSF73-II, inhibiting growth through coordinating gene expression and poly(A) site choice. Functional enrichment analysis revealed that the accumulation of ethylene and auxin jointly inhibited primary root growth. AN3661 affected poly(A) signaling recognition, resulted in lower U-rich signal usage, caused transcriptional readthrough, and increased the distal poly(A) site usage. Many microRNA targets were found in the 3' UTR lengthened transcripts; these miRNAs may indirectly regulate the expression of these targets through cleavage. Overall, this work demonstrates that AtCPSF73 plays important parts in co-transcriptional regulation, affecting growth and development in Arabidopsis.