Tandem 3′ UTR events exhibited an even higher frequency of tissue-regulated expression than SEs or other AS events studied (), yet little is known about how tissue regulation of tandem UTRs is accomplished, e.g., whether through APA or through differential stability of alternative UTR isoforms. Grouping tandem 3′ UTRs by switch score, the most switch-like events exhibited increased sequence conservation relative to those with lower switch scores in the vicinity of and upstream of the proximal (5′) polyadenylation signal (PAS), and also upstream of the distal (3′) PAS (). While cis-regulatory elements contributing to differential stability should be located predominantly in the region unique to the long UTR isoform, APA could be regulated by elements located near either or both PAS. The observation of increased conservation around and upstream of the proximal PAS in switch-like tandem UTRs therefore supports a primary role for regulation at the level of APA.
Evidence for coordination between splicing and polyadenylation
In assessing the spectrum of cis
-elements that may drive tissue regulation of tandem 3′ UTRs, a set of 7mers was identified that exhibited high conservation in the extension region of tandem 3′ UTRs (), with signal:background (S:B) ratios in 4 mammals26
exceeding 2:1. As expected, this set included the extended (7-base) seed matches to a number of conserved mammalian miRNAs26-28
. Surprisingly, it also included all 8 of the 7mers that contain the Fox-1/-2 consensus binding motif, UGCAUG: all such 7mers had S:B ratios above 2.5:1, exceeding the S:B observed for seed matches to important miRNAs such as miR-7 and miR-181 (inset, ). Strong conservation of UGCAUG motifs in this location (>1 kbp on average from the nearest splice site) would not be expected on the basis of the canonical splicing regulatory activity of Fox-1/Fox-2 proteins. Instead, the high conservation observed in extended 3′ UTR regions suggests that these factors (or others with identical RNA binding specificity) play additional 3′ UTR-related roles, e.g., in APA, or in mRNA localization and/or translation.
To further investigate possible connections between tissue-specific regulation of AS and APA, global patterns of tissue-specific alternative isoform expression were compared. Applying singular value decomposition (SVD) (Supplemental Methods
) to the vectors of inclusion ratios across samples for each AS and APA event type separately, a strong and consistent separation of the breast cell lines (4 cancer-derived and one immortalized cell line) from all tissue samples was observed (). This separation implied the existence of a systematic difference in RNA processing regulation between cell lines and tissues that held for all types of AS events studied. For most AS and APA events, SVD analysis yielded similar groupings of tissues, e.g., with heart, skeletal muscle, brain and liver consistently clustered (Fig. S8
). Consistent with this observation, pairwise distances between SVD projections for different types of AS events, e.g., SE, A5SS, and A3SS were all highly correlated (), suggesting similarities in the regulatory control of these types of events1,2,13
. More surprisingly, distances between SVD projections for tandem 3′ UTR events also correlated highly with those for events controlled purely at the level of splicing such as SEs (). This observation raised the possibility that splicing and polyadenylation may be coordinately regulated across human tissues.
To explore possible regulatory connections between splicing and polyadenylation regulation (e.g., refs29-32
), the conservation of 6mers adjacent to conserved AS and APA events was compared. While canonical 3′ UTR regulatory motifs such as the consensus PAS 6mer AAUAAA and various miRNA seed matches exhibited high S:B ratios, often 1.5:1 or higher, in extended 3′ UTR regions, these motifs generally had S:B ratios close to 1:1 in AS introns. However, a distinct subset of motifs with high S:B ratios in both UTRs and introns was also observed, several of which corresponded to well-known splicing-related motifs (, Table S8
). This set included not only the Fox-1/-2 motif UGCAUG and variations, consistent with the 7mer analysis of , but also permutations of (CUG)n
, which represent putative substrates of the CELF and MBNL families of muscle- and brain-specific splicing factors33
. The highly significant S:B in both 3′ UTRs and introns suggested that these well known splicing-related motifs also commonly play 3′ UTR-related roles – e.g., control of APA or of mRNA stability, localization, or translation – as recently demonstrated for the Nova family of splicing factors34
The 6mer ACUAAC, an excellent match to the consensus binding motifs of STAR family RNA binding factors, in particular Quaking/QKI35
, was also notable. Not only did ACUAAC have significant S:B in 3′ UTRs, as expected from QKI's known role in control of mRNA stability36
, but it also showed extremely high S:B in introns, exceeding 7:1. This extreme conservation suggested a common and important function in splicing regulation, a role which has been suggested but not yet directly demonstrated9,37
. Motif enrichment analyses also suggested a possible role in brain-specific APA regulation (Fig. S9