Analysis of circadian regulation of poly(A)-tail length

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 posttranscriptionally 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 depending on differential fractionation of mRNAs depending on the length of their tails has recently been developed. In this chapter, we describe these methods as used for examining the circadian regulation of poly(A)-tail length and 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.