Heterogeneity in polyadenylation cleavage sites in mammalian mRNA sequences: implications for SAGE analysis

Abstract

The analysis of a human thyroid serial analysis of gene expression (SAGE) library shows the presence of an abundant SAGE tag corresponding to the mRNA of thyroglobulin (TG). Additional, less abundant tags are present that can not be linked to any other known gene, but show considerable homology to the wild-type TG tag. To determine whether these tags represent TG mRNA molecules with alternative cleavage, 3′-RACE clones were sequenced. The results show that the three putative TG SAGE tags can be attributed to TG transcripts and reflect the use of alternative polyadenylation cleavage sites downstream of a single polyadenylation signal in vivo. By screening more than 300 000 sequences corresponding to human, mouse and rat transcripts for this phenomenon we show that a considerable percentage of mRNA transcripts (44% human, 22% mouse and 22% rat) show cleavage site heterogeneity. When analyzing SAGE-generated expression data, this phenomenon should be considered, since, according to our calculations, 2.8% of human transcripts show two or more different SAGE tags corresponding to a single gene because of alternative cleavage site selection. Both experimental and in silico data show that the selection of the specific cleavage site for poly(A) addition using a given polyadenylation signal is more variable than was previously thought.

Long-Range Heterogeneity at the 3′ Ends of Human mRNAs

“…This "clean" set was then mapped onto the genome sequences and genomelinked tags were derived by combining the 50 nt 3Ј tag with the 50 nt following the tag in the genome (the total genomelinked tag length is thus 100 nt). These combined tags were clustered to resolve short-range variations in the exact polyadenylation site between individual transcript sequences; it should be noted that this procedure eliminates from our analysis the short-range variation shown by Pauws et al (2001). After clustering, 13% of the tags identified genome regions with nonidentical downstream sequences.…”

“…Although there have been numerous reports in the literature of transcripts undergoing alternative polyadenylation involving relatively distant sites (van Eyndhoven et al 1998;Coy et al 1999;Touriol et al 1999), it was unexpected to observe it at this high frequency. Estimates gathered from EST clustering alone (Gautheret et al 1998;Beaudoing and Gautheret 2001;Pauws et al 2001). We experimentally verified our methodology by performing reverse transcriptasepolymerase chain reaction (RT-PCR) experiments on the predicted long 3Ј UTRs of the WDR9 (WD repeat 9) and KCNJ5 (potassium inwardly rectifying channel 5) genes and found that they do indeed encode transcripts extending far downstream from the 3Ј ends documented by "full-length" cDNA sequences, even though the intervening regions are not fully covered by ESTs (data not shown).…”

“…The collection of trusted 3Ј tags and the assignment of associated EST clusters to the 3Ј UTR of validated genes will thus be a crucial resource for the rational design of hybridization arrays. Another important implication of the work described here is the need for more comprehensive tag to gene maps for SAGE experiments (Pauws et al 2001). Current maps do not take into account the locations of trusted mRNA 3Ј ends.…”

“…Of the three major mechanisms that contribute to this complexity, alternative initiation of transcription, splicing, and polyadenylation, the latter seemed most immediately amenable to analysis because of the wealth of data about transcript 3Ј ends provided by the expressed sequence tag (EST) sequences generated by the NCI Cancer Genome Anatomy Project (Strausberg et al 2000) (at Washington University, the NIH Intramural Sequencing Center, and Incyte Pharmaceuticals), the Merck Gene Index (Aaronson et al 1996), and the NIH Mammalian Gene Collection (Strausberg et al 1999). Although alternative polyadenylation of transcripts has been known to occur for a long time, the proportion of transcripts affected, the number of sites per transcript, and the distances over which alternative sites are spread have been explored exclusively using EST clustering techniques (Gautheret et al 1998;Beaudoing and Gautheret 2001;Pauws et al 2001) and have relied on the poly(A) being documented in the EST sequences. The most recent of these studies have concluded that >40% of human transcripts may undergo alternative polyadenylation, but that most of the observed variation is over a short range (<50 nt) and driven by a single polyadenylation signal (Beaudoing and Gautheret 2001;Pauws et al 2001).…”

“…Although alternative polyadenylation of transcripts has been known to occur for a long time, the proportion of transcripts affected, the number of sites per transcript, and the distances over which alternative sites are spread have been explored exclusively using EST clustering techniques (Gautheret et al 1998;Beaudoing and Gautheret 2001;Pauws et al 2001) and have relied on the poly(A) being documented in the EST sequences. The most recent of these studies have concluded that >40% of human transcripts may undergo alternative polyadenylation, but that most of the observed variation is over a short range (<50 nt) and driven by a single polyadenylation signal (Beaudoing and Gautheret 2001;Pauws et al 2001). Long-range variation (>1 kb) has so far been observed only experimentally.…”