This is a follow-up of sorts to a previous essay on the subject of alternative polyadenylation. In the previous report, I discussed some bioinformatics studies that suggested that the 3′ UTRs of mRNAs change, in bulk, in the course of development in mammals. The implication of these results is that poly(A) site choice in mammals is regulated, with important functional consequences.
A more recent study by Mayr and Bartel adds to this notion. These authors studied 3′ UTR length in normal and cancer cells, and found a striking correlation between 3′ UTR length and the expression of oncogenes. Specifically, higher expression (as is found in cancer cells) is correlated with shorter 3′ UTR. As 3′ UTR length is determined by the position of the poly(A) site along a transcript, this implicates alternative polyadenylation as one mechanism by which oncogene expression is activated.
In cancer cells, genetic alterations can activate proto-oncogenes, thereby contributing to tumorigenesis. However, the protein products of oncogenes are sometimes overexpressed without alteration of the proto-oncogene. Helping to explain this phenomenon, we found that when compared to similarly proliferating nontransformed cell lines, cancer cell lines often expressed substantial amounts of mRNA isoforms with shorter 3′ untranslated regions (UTRs). These shorter isoforms usually resulted from alternative cleavage and polyadenylation (APA). The APA had functional consequences, with the shorter mRNA isoforms exhibiting increased stability and typically producing ten-fold more protein, in part through the loss of microRNA-mediated repression. Moreover, expression of the shorter mRNA isoform of the proto-oncogene IGF2BP1/IMP-1 led to far more oncogenic transformation than did expression of the full-length, annotated mRNA. The high incidence of APA in cancer cells, with consequent loss of 3′UTR repressive elements, suggests a pervasive role for APA in oncogene activation without genetic alteration.
These authors studied a selected group of genes using RNA blotting (northern blotting). The selected genes had a good predicted potential for alternative polyadenylation, and were possible microRNA targets. The idea is that oncogene expression may be increased by eliminating microRNA target sequences within 3′-UTRs, via alternative polyadenylation. Indeed, the authors found that the subjects of the study had shorter 3′ UTRs in cancer cells, and that the shorter mRNAs were both more stable and translated more efficiently. These latter features correlated with microRNA targeting. (Recall that mRNA stability and translational repression are the two principle mechanisms by which microRNAs control gene expression.)
So, from the perspective of polyadenylation, what is going on? There is no clear answer, but the authors speculate as to posisble regulatory mechanisms:
One of the most interesting open questions regarding APA in cancer cells is, what mechanism underlies the recognition and increased utilization of proximal polyadenylation signals? In the sequence surrounding the proximal polyadenylation sites we never found point mutations that would have changed the strength of the polyadenylation signal, with the caveat that by 3′ RACE we investigated only the sequences upstream of the cleavage sites. Although mutations downstream of the cleavage sites cannot be excluded, we hypothesize that differential expression of trans-acting factors explains the use of proximal polyadenylation sites in cancer cells. Factors that might influence the choice of polyadenylation signal include limiting components of the polyadenylation machinery, RNA-binding proteins that bind in the vicinity of the proximal signal and influence recognition by the polyadenylation machinery ([Takagaki et al., 1996], [Martincic et al., 1998], [Veraldi et al., 2001], [Lutz, 2008] and [Wang et al., 2008]), and perhaps factors that influence transcriptional elongation rate (Kornblihtt, 2005). To begin to identify such factors, we examined published array data comparing breast cancer cells with a breast epithelial cell line, MCF10A (Hoeflich et al., 2009). A survey of the constitutive components of the polyadenylation machinery and other candidates from the literature revealed several that were significantly upregulated in the cancer lines (Figure S8). These included the mRNAs of cleavage and polyadenylation specificity factor 1 (CPSF1) and cleavage stimulation factor 2 (CSTF2), which recognize the poly(A) signal and accessory sequences including the downstream G/U-rich sequence, respectively, raising the intriguing possibility that an increase of these factors might help increase utilization of sub-optimal proximal poly(A) signals in cancer cells.
We imagine a complex scenario in which some trans-acting factors act globally, some act tissue specifically, and some act gene specifically, with the combinatorial expression of all the different trans-acting factors determining the probability of using each proximal polyadenylation signal. The observation of higher amounts of shorter mRNAs in cancer cells compared with normal cells, with no examples of the reverse for any of the genes and cell types studied, suggested a role for globally acting factors. That some cell lines showed high amounts of shorter transcripts for all genes investigated further implicated the role of globally acting factors. Nonetheless, the differences observed for different genes in different cell types suggested a role for additional factors acting more specifically. Such complexity could explain the differential impact of different oncogenes in different tissues. Indeed, some of the genes for which we did not observe alternative mRNAs are known oncogenes in tissues not included in our panel of cell lines (ARID3B in neuroblastomas, MYB and PLAG1 in hematological malignancies). Perhaps shorter mRNA transcripts might be found in the tissues where these genes have oncogenic effects. Moreover, the prospect of some factors acting more specifically opens the possibility for exceptions to the trend of shorter isoforms expressed preferentially in cancer cells. Combinatorial use of tissue-specific and gene-specific trans-acting factors could for some genes (most intriguingly, for tumor-suppressor genes) lead to higher amounts of shorter mRNAs in normal cells rather than in cancer cells.
Clearly, much remains to be learned. However, the ramifications as far as the control of the polyadenylation complex are exciting.