Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs

BackgroundShort interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels.ResultsHere, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels.ConclusionsOur study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1083-0) contains supplementary material, which is available to authorized users.

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“…The length of the poly(A) tail was determined by PCR following a splint-linker ligation as described [49]. …”

Section: Methodsmentioning

confidence: 99%

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity

et al. 2016

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“…In the structure, GLD-3 covers hydrophobic surface patches of GLD-2, shielding them from solvent. With hindsight, the finding that GLD-2 in isolation is capable of adding only a few adenosines (10,23,43) (Fig. 1B) might be explained by the rapid inactivation of the protein in the time and conditions of the assays.…”

Section: Gld-3 Contributes Both Directly and Indirectly To The Enzymaticmentioning

confidence: 99%

Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3

Nakel

Bonneau

Eckmann

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The Caenorhabditis elegans germ-line development defective (GLD)-2–GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2–GLD-3 acts by extending the short poly(A) tail of germ-line–specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3′ end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3′ end features of dormant cytoplasmic mRNAs.

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“…More recent techniques are all based on RT-PCR, rather than Northern Blot, and successfully overcome these issues by providing increased sensitivity, speed and length quantification. Several methods, such as RACE-PAT (Salles, Darrow, O'Connell, & Strickland, 1992), and Ligation-Mediated (LM)-PAT (Salles & Strickland, 1995), extension PAT (ePAT) (Janicke, Vancuylenberg, Boag, Traven, & Beilharz, 2012) or splint-mediated PAT (sPAT) (Minasaki, Rudel, & Eckmann, 2014), have been described, and these are slightly different in their technology in the cDNA synthesis step, each having advantages and disadvantages.…”

Section: Measurement Of Poly(a) Tail Length At a Single-gene Levelmentioning

confidence: 99%

Analysis of Circadian Regulation of Poly(A)-Tail Length

Kojima

Green

2015

Circadian Rhythms and Biological Clocks, Part A

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The poly(A) tail is found on the 3’-end of most eukaryotic mRNAs, and its length significantly contributes to the mRNAs half-life and translational competence. Circadian regulation of poly(A) tail length is a powerful mechanism to confer rhythmicity in gene expression post-transcriptionally, and provides a means to regulate protein levels independent of rhythmic transcription in the nucleus. Therefore, analysis of circadian poly(A) tail length regulation is important for a complete understanding of rhythmic physiology, since rhythmically expressed proteins are the ultimate mediators of rhythmic function. Nevertheless, it has previously been challenging to measure changes in poly(A) tail length, especially at a global level, due to technical constraints. However, new methodology based on differential fractionation of mRNAs based on the length of their tails has recently been developed. In this chapter, we will describe these methods as used for examining the circadian regulation of poly(A) tail length and will provide detailed experimental procedures to measure poly(A) tail length both at a the single mRNA level and the global level. Although this chapter concentrates on methods we used for analyzing poly(A) tail length in the mammalian circadian system, the methods described here can be applicable to any organisms and any biological processes.

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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: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs, RNA Biology, 11:2, 111-123,

Cited by 16 publications

References 43 publications

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity

Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3

Analysis of Circadian Regulation of Poly(A)-Tail Length

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