Tales of Detailed Poly(A) Tails

Abstract

Poly(A) tails are non-templated additions of adenosines at the 3' ends of most eukaryotic mRNAs. In the nucleus, these RNAs are co-transcriptionally cleaved at a poly(A) site and then polyadenylated before being exported to the cytoplasm. In the cytoplasm, poly(A) tails play pivotal roles in the translation and stability of the mRNA. One challenge in studying poly(A) tails is that they are difficult to sequence and accurately measure. However, recent advances in sequencing technology, computational algorithms, and other assays have enabled a more detailed look at poly(A) tail length genome-wide throughout many developmental stages and organisms. With the help of these advances, our understanding of poly(A) tail length has evolved over the past 5 years with the recognition that highly expressed genes can have short poly(A) tails and the elucidation of the seemingly contradictory roles for poly(A)-binding protein (PABP) in facilitating both protection and deadenylation.

Keywords: deadenylation; poly(A) tail; poly(A)-binding protein; translation.

Figure 1:

Comparison of different sequencing methods for reading poly(A) tails. A) TAIL-seq is able to capture the 3’ end of any RNA, and therefore gives a readout of both poly(A) tail length as well as other modifications such as uridylation through an innovative Tailseeker algorithm. B) PAL-seq uses a splint oligo to preferentially capture polyadenylated RNAs, thus bypassing rRNA removal. Biotin-labeled dUTP marks each cluster in proportion to the length of the tail. C) mTAIL-seq uses the splint oligo approach in order to reduce the amount of starting material needed, and uses the Tailseeker software to read poly(A) tail length. D) Nanopore technology is a new way to sequence that can be used to directly sequence RNA or cDNA with minimal library preparation needed. The nucleic acid travels through the nanopore at a constant rate; therefore, the dwell time of the poly(A) tail in the nanopore correlates to its length.

Figure 2:

Short poly(A) tails are associated with highly expressed, well-translated transcripts. These shortened tails occur at discrete lengths that have a phasing pattern matching the footprint of serial binding of cytoplasmic poly(A) binding protein. Longer tails do not show this phasing and have less well-defined tails. In somatic cells, short tailed transcripts tend to have high codon optimality and long half lives.

Figure 3:

Differential activities of the Ccr4 and Caf1 deadenylases. A) Caf1 is able to deadenylate portions of the poly(A) tail that are not tightly bound by cytoplasmic poly(A) binding protein (PABPC) but will halt once it reaches PABPC. On the other hand, Ccr4 is able to displace PABPC from the poly(A) tail and continue deadenylating. Ccr4 is also able to act on poly(A) stretches that are not bound by PABPC. B) Caf1 preferentially accelerates deadenylation of low codon optimality transcripts. Ccr4 is able to act on both substrates but high codon optimality transcripts seem to rely solely on Ccr4-mediated deadenylation. This may be due to differences in PABPC occupancy on the tails of transcripts with higher (more PABPC) and lower (less PABPC) levels of optimal codons.