Multiple Mechanisms Driving Alternative Polyadenylation of Cyclin D1 (CCND1) pre‐mRNA Processing

Abstract

Eukaryotic 3′end formation involves binding of 3′end processing factors to the pre‐mRNA, cleavage downstream of a polyadenylation signal (PAS), followed by polyadenylation. The existence of multiple PASs in ~70% of human genes has led to the increased recognition of alternative polyadenylation (APA) as a new paradigm of global gene regulation involving the 3′end. Transcripts resulting from APA of the same terminal exon share the same open reading frame but have 3′UTRs of different length which affect the stability, localization and translatability of the mRNA transcript. One of the genes that undergoes APA is cyclin D1 (CCND1), which is rate limiting for G1‐S phase cell cycle transition. Cyclin D1 expression is tightly regulated and is normally transiently expressed in response to mitogenic stimuli. Both the normal protein and full length CCND1 mRNA transcript have a short half–life (~30 minutes). It is therefore not surprising that cyclin D1 expression is deregulated in several pathological states including cancer. In the B‐cell malignancy mantle cell lymphoma (MCL), the initiating lesion is a t(11; 14) (q13; q32) chromosomal translocation event that juxtaposes the CCND1 gene downstream of the IgH locus, resulting in constitutive expression of cyclin D1. Some MCL patients express CCND1 transcripts with truncated 3′UTRs and this is associated with a decrease in survival of almost two years. The truncated CCND1 transcript eliminates ~ 3kb from the 3′UTR leaving a 3′UTR <0.5kb in length. Our goal is to determine the mechanisms involved in APA as well as develop tools to identify and detect changes in the 3′UTR of genes. We developed different qRT‐PCR primers that can detect CCND1 transcripts with long/short 3′UTRs as well as nested primers for 3′RACE to help map the 3′ends of different CCND1 transcripts. In addition, used Illumina HiSeq and Pacific Biosciences (PacBio) next generation sequencing platforms to detect alternative transcripts. Reporter plasmids containing 3′UTRs were used to determine effects of RNAi. We found that highly proliferative cell lines were associated with higher levels of truncated CCND1 3′UTRs. One cell line had a mutation that resulted in the generation of a canonical PAS from a non‐optimal PAS proximal to the stop codon causing a switch from using a more distal PAS to the mutated PAS proximal to the stop codon. Illumina transcriptome sequencing after knockdown of a 3′end processing factor, CFIm25, resulted in increased shortening of the 3′UTR of CCND1 and over 1,400 other genes. Hence APA of CCND1 may be regulated by altered expression/function of genes involved in 3′end formation. When we mapped the 3′ends of CCND1 transcripts from multiple cell lines using 3′RACE, we identified a novel CCND1 transcript with a chimeric 3′UTR consisting of sequences from CCND1 fused to another gene, MCRK. This transcript containing the chimeric CCND1‐MRCK sequence was only detected in some MCL cell lines and patient DNA. Full length CCND1 3′UTR has over 60 potential miRNA target sites and reporter assays showed that the CCND1‐MRCK 3′UTR was able to evade miRNA regulation. Preliminary full length transcript PacBio sequencing resulted in the de novo identification of the CCND1‐MRCK fusion transcript. In conclusion, there are multiple ways in which CCND1 optimizes its expression in cells at the mRNA level and these mechanisms may be used by other genes under different physiological states.Support or Funding InformationDoD W81XWH‐12‐1‐0218This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.