Fear and polyadenylation

No, this post is not about the fear that our favorite subject strikes in the minds of students who are struggling with concepts and principles of gene expression.  Rather, it’s about an interesting story that helps to illustrate (as if this is needed) the relevance of polyadenylation (and specifically poly(A) site choice) to medical science.

Mention has been made on this blog of a correlation between poly(A) site choice and cancer.  Many meta-analyses and high throughput sequencing studies have also noted a related phenomenon – a great deal of alternative polyadenylation that seems to be specific for neural cells and tissues.  One example of this is recalled in a recent paper that suggests a link between an alteration in alternative polyadenylation and aspects of memory and anxiety in mammals (including humans).

This study (and an earlier one from the same group that touches on the subject) traces its way back to a report from 1999 about a poly(A) site polymorphism in the human gene for the serotonin transporter (hSERT, or 5-HTT).  There were two findings in this study that factor into the more recent findings:  transcripts encoded by the hSERT gene could be polyadenylated at either of two sites, and there was a polymorphism seen in individuals that lies within the putative poly(A) signal for the distal site.  This is shown in Figure 2 of the 1999 paper (reproduced here, from the journal web site, with apologies for the somewhat poor quality of the reproduction):

In this summary, the locations of the two poly(A) sites are denoted by the “-[poly A]” notation, the associated poly(A) signals are underlined, and the nature of the G/T polymorphism in the distal signal indicated.

In the two later studies, the correlation of the G/T polymorphism and behavioral disorders in humans was studied.  These genetic studies were accompanied by experiments with 5-HTT knock-out mice, so as to corroborate the associations seen in the genetic studies.  The abstract of the 2010 paper (Gyawali et al.) states:

Background: Genetic markers in the serotonin transporter are associated with panic disorder (PD). The associated polymorphisms do not include the serotonin transporter-linked polymorphic region and display no obvious functional attributes. A common polymorphism (rs3813034) occurs in one of the two reported polyadenylation signals for the serotonin transporter and is in linkage disequilibrium with the PD-associated markers. If functional, rs3813034 might be the risk factor that explains the association of the serotonin transporter and PD.

Methods: Quantitative polymerase chain reaction on human brain samples (n = 65) and lymphoblast cultures (n = 71) was used to test rs3813034 for effects on expression of the polyadenylation forms of the serotonin transporter. rs3813034 was also tested for association in a sample of PD cases (n = 307) and a control sample (n = 542) that has similar population structure.

Results: The balance of the two polyadenylation forms of the serotonin transporter is associated with rs3813034 in brain (p < .001) and lymphoblasts (p < .001). The balance of the polyadenylation forms is also associated with gender in brain only (p < .05). Association testing of rs3813034 in PD identified a significant association (p < .0068) with a relative risk of 1.56 and 1.81 for the heterozygous and homozygous variant genotypes, respectively.

Conclusions: rs3813034 is a functional polymorphism in the serotonin transporter that alters the balance of the two polyadenylation forms of the serotonin transporter. rs3813034 is a putative risk factor for PD and other behavioral disorders that involve dysregulation of serotonergic neurotransmission.

The abstract from the more recent (Hartley et al., 2012) paper:

Growing evidence suggests serotonin’s role in anxiety and depression is mediated by its effects on learned fear associations. Pharmacological and genetic manipulations of serotonin signaling in mice alter the retention of fear extinction learning, which is inversely associated with anxious temperament in mice and humans. Here, we test whether genetic variation in serotonin signaling in the form of a common human serotonin transporter polyadenylation polymorphism (STPP/rs3813034) is associated with spontaneous fear recovery after extinction. We show that the risk allele of this polymorphism is associated with impaired retention of fear extinction memory and heightened anxiety and depressive symptoms. These STPP associations in humans mirror the phenotypic effects of serotonin transporter knockout in mice, highlighting the STPP as a potential genetic locus underlying interindividual differences in serotonin transporter function in humans. Furthermore, we show that the serotonin transporter polyadenylation profile associated with the STPP risk allele is altered through the chronic administration of fluoxetine, a treatment that also facilitates retention of extinction learning. The propensity to form persistent fear associations due to poor extinction recall may be an intermediate phenotype mediating the effects of genetic variation in serotonergic function on anxiety and depression. The consistency and specificity of these data across species provide robust support for this hypothesis and suggest that the little-studied STPP may be an important risk factor for mood and anxiety disorders in humans.

Interestingly, another study reports that SERT transcripts are targets of a microRNA, miR-16:

I’ve taken the liberty of summarizing all of this:

In this figure, the two poly(A) signals are in underlined italicized lettering, the two poly(A) sites are in bold underlined lettering, and the possible miR-16 homologies complementarities are underlined.  The miR-16 sequence (read 3′-5′ to make it easier to compare with the above) is FYI:

3′-GCGGUUAUAAAUGCACGACGAU-5′

(Note that U-T in the corresponding DNA sequence, and that it is necessary to write out the homologous sequence to compare with my sequence rendition.  Also, note that the miR16 homology described in 1999 is the promoter-proximal site I underline in my summary; I have taken the liberty of underlining a second possible target site, albeit one that disobeys the rules for microRNA target recognition.)

A few notes about all of this:

1.  In the “normal” gene, the distal poly(A) signal is AUUAAC, whereas in the panid disorer (PD)-associated variant, it is AGUAAC.  The U->G alteration is associated with reduced distal poly(A) site usage, as might be expected if AUUAAC were the poly(A) signal for the distal site.

2.  The correlation between poly(A) site choice and behavior seems to be well-supported by the data.  However, neither putative poly(A) signal is the canonical mammalian AAUAAA; the signal for the shorter transcript is AAUGAA, a close match but still usually thought of as sub-optimal, while the putative signal for the longer transcript, AUUAAC, is quite different from what is normally thought of as a poly(A) signal.  There is a possible signal (AAUAUA) situated upstream of this, but it is farther than usual from the poly(A) site itself.  I believe the authors wish to link the behavior-associated polymorphism with a poly(A) signal (in this case, the “normal” allele AUUAAC and the variant, AGUAAC, that may be a risk determinant for panic disorder), but it may be that the story here is more complicated.

3.  It’s not clear how the putative miR16 target site factors into all of this.  The site highlighted in Baudry et al. lies upstream from the promoter-proximal site, and thus should be in both transcripts.  There is another extended homology that lies between the two sites, but the sequence matches are at the “wrong” end of the microRNA (see this essay for an explanation).

4.  It’s interesting that one of the apparent targets of fluoxetine (=Prozac) is the choice of poly(A) site in this gene; specifically, Prozac seems to promote distal poly(A) site choice.

5.  It’s also interesting that there is a gender difference in distal poly(A) site utilization in this gene.

The reading list for this essay:

Battersby S, Ogilvie AD, Blackwood DH, Shen S, Muqit MM, Muir WJ, Teague P, Goodwin GM, Harmar AJ. Presence of multiple functional polyadenylation signals and a single nucleotide polymorphism in the 3′ untranslated region of the human serotonin transporter gene. J Neurochem. 1999 Apr;72(4):1384-8. PubMed PMID: 10098839.

Baudry A, Mouillet-Richard S, Schneider B, Launay JM, Kellermann O. miR-16 targets the serotonin transporter: a new facet for adaptive responses to antidepressants. Science. 2010 Sep 17;329(5998):1537-41. PubMed PMID: 20847275.

Gyawali S, Subaran R, Weissman MM, Hershkowitz D, McKenna MC, Talati A, Fyer AJ, Wickramaratne P, Adams PB, Hodge SE, Schmidt CJ, Bannon MJ, Glatt CE. Association of a polyadenylation polymorphism in the serotonin transporter and panic disorder. Biol Psychiatry. 2010 Feb 15;67(4):331-8. Epub 2009 Dec 6. PubMed PMID: 19969287; PubMed Central PMCID: PMC2980348.

Hartley CA, McKenna MC, Salman R, Holmes A, Casey BJ, Phelps EA, Glatt CE. Serotonin transporter polyadenylation polymorphism modulates the retention of fear extinction memory. Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5493-8. Epub 2012 Mar 19. PubMed PMID: 22431634; PubMed Central PMCID: PMC3325655.