Unlike EST based approaches, these methods based on 3P-Seq precisely detect the usage of different 3 ′ UTRs in mRNAs.

Using 3P-Seq data, the 3 ′ UTRs of genes in yeast, worm, fly, zebrafish, mouse and human genomes have been annotated in ( Andrew et al., 2010 Derti et al., 2012 Hoque et al., 2013 Jan et al., 2011 Mangone et al., 2010 Nam et al., 2014 Shepard et al., 2011 Ulitsky et al., 2012). However, an EST based approach is not able to estimate the relative abundance of each 3 ′ UTR in the resultant mRNAs ( Kim et al., 2015b). Not only does this analysis show that mammalian genes have multiple 3 ′ UTRs ( Tian, 2005), but also it reveals that neuronal cell mRNAs have longer 3 ′ UTRs than liver cell mRNAs ( Sood et al., 2006). The analysis of expressed sequence tags (ESTs) has provided genome-wide annotations of 3 ′ UTRs. Therefore, the analysis of alternative (or all) polyadenylation sites (APA sites) would be of great importance for the study of mammalian genes. According to the study in ( Christine, 2016), more than half of human genes have alternative polyadenylation in their post-transcriptional process. Alternative polyadenylation (cleavage) is very common in mammalian genes ( Tian, 2005). Moreover, the secondary structure of a 3 ′ UTR is also important for its translation efficiency and disruption of expression ( Barrett et al., 2012). The length of a 3 ′ UTR as well as some sequence elements in the 3 ′ UTR such as AU-rich elements and GU-rich elements may have impact on mRNA stability, mRNA localization, protein translation, protein binding and translation efficiency ( Barrett et al., 2012). Note that a 3 ′ UTR is a suffix of an mRNA sandwiched between the stop codon and polyadenylation cleavage site of the mRNA. Due to the interactions between these cis elements and polyadenylation factors, alternative cleavage sites can be formed for a pre-mRNA, resulting in more than one mRNA transcript from a single pre-mRNA containing 3 ′ untranslated regions ( ⁠ 3 ′ UTRs) of different lengths. In addition, some auxiliary elements upstream of the PAS and downstream of the cleavage site may enhance the polyadenylation process ( Tian, 2005). These U-rich or U/G-rich elements serve as the binding sites for the cleavage stimulation factor (CstF). U-rich or U/G-rich elements located 20–40 bps downstream of the cleavage site are also involved in polyadenylation ( Tian, 2005). The PAS serves as a binding site for the cleavage and polyadenylation specificity factor (CPSF). This hexamer is called the polyadenylation signal (PAS) and it usually appears 10–30 bps upstream of the cleavage site ( Tian, 2005).

In particular, the 3 ′ end sequence of a pre-mRNA usually contains a AAUAAA hexamer (or some close variant). Certain cis-acting elements and trans-acting factors have been found in the literature that influence the choice of a particular polyA cleavage site ( Barrett et al., 2012 Pichon et al., 2012). More precisely, the polyadenylation process consists of two steps ( Wahle and Kühn, 1997): cleavage near the 3 ′ end of a pre-mRNA and the addition of a polyA tail at the cleavage site. In particular, a polyA tail is added at the 3 ′ end of a pre-mRNA with the help of the polyadenylation process. The post-transcriptional process includes three major steps: the addition of a 5 ′ cap, addition of a polyadenylation (polyA) tail and splicing. Finally, this mature mRNA is translated into the corresponding protein. The pre-mRNA is then converted to a mature mRNA by the post-transcriptional process. Furthermore, the tools are unable to integrate the analysis of shortening/lengthening events with APA site detection.Īccording to the central dogma of molecular biology, the transcription process in eukaryotes synthesizes a pre-mRNA from the genomic sequence of a gene ( Leavitt and Nirenberg, 2010). These tools consider either up to only two APA sites in a gene or only APA sites that occur in the last exon of a gene, although a gene may generally have more than two APA sites and an APA site may sometimes occur before the last exon. As alternative polyadenylation (APA) sites are common in mammalian genes, several tools have been published recently for detecting APA sites from RNA-Seq data or performing shortening/lengthening analysis. This length is largely determined by the polyadenylation cleavage site in the mRNA. Moreover, correlation between diseases and the shortening (or lengthening) of 3 ′ UTRs has been reported in the literature. The length of the 3 ′ untranslated region ( ⁠ 3 ′ UTR) of an mRNA is essential for many biological activities such as mRNA stability, sub-cellular localization, protein translation, protein binding and translation efficiency.

Alternatives siteanalyzer