title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs.
abstract: Poly(A) tail length is a readout of an mRNA's translatability and stability, especially in developmental systems. PolyAdenylation Test (PAT) assays attempt to quickly measure the average poly(A) tail length of RNAs of experimental interest. Here we present sPAT, splint-mediated PAT, a procedure that uses a DNA splint to aid in the ligation of an RNA-tag to the poly(A) tail of an mRNA. In comparison to other PAT methodologies, including ePAT, sPAT is highly sensitive to low-abundance mRNAs, gives a more accurate profile of the poly(A) tail distribution, and requires little starting material. To demonstrate its strength, we calibrated sPAT on defined poly(A) tails of synthetic mRNAs, reassessed developmentally regulated poly(A) tail-length changes of known mRNAs from established model organisms, and extended it to the emerging evolutionary developmental nematode model Pristionchus pacificus. Lastly, we used sPAT to analyze the contribution of the two cytoplasmic poly(A) polymerases GLD-2 and GLD-4, and the deadenylase CCR-4, onto Caenorhabditis elegans gld-1 mRNA that encodes a translationally controlled tumor suppressor whose poly(A) tail length measurement proved elusive.
authors: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG); Pfotenhauerstrasse 108; 01307 Dresden, Germany.
authors: Ryuji
authors: R
authors: Minasaki
authors: Department of Biology; East Carolina University; Greenville, NC USA.
authors: David
authors: D
authors: Rudel
authors: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG); Pfotenhauerstrasse 108; 01307 Dresden, Germany.
authors: Christian R
authors: CR
authors: Eckmann
mesh_terms: D000818:Animals; D004277:DNA, Single-Stranded / Q000378:metabolism*; D019143:Evolution, Molecular; D005821:Genetic Techniques; D023421:Models, Animal; D010802:Phylogeny; D011061:Poly A / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D011061:Poly A
keywords: CCR-4; GLD-2; GLD-4; deadenylases; ePAT; gld-1; poly(A) polymerases; poly(A) tail; sPAT