The circadian photoneuroendocrine system consists of the retina, suprachiasmatic nucleus, and the pineal gland (Rhode 2014). It transforms external light captured by the photoreceptors of the retina into the internal melatonin signal secreted by the pineal gland in the brain (Kovacs and Ojeda 2012; Rhode 2014). Usually, melatonin secretion increases in the evening and decreases in the morning (Cutolo et al. 2003; Rhode 2014). Aanat is in involved in the rate-limiting step of the biosynthetic pathway that converts serotonin to melatonin (Roseboom 1998; Sakamoto and Ishida 1998).
Due to repeated inbreeding, wild type C57BL/6J mice are homozygous for a SNP within the Aanat gene, that is absent in wild type 129/SV mice. The guanine-to-adenine base difference in the third intron generates a potentially new splice acceptor site in the C57BL/6J mice (Fig. 1). The presence of this nucleotide difference may result in an alternative RNA splicing process in the C57BL/6J mice. The inclusion of the pseudo-exon introduces a premature stop codon, resulting in the generation of a truncated and non-functional protein (<1% of proper enzymatic activity).
The goal of this project is to confirm the genotypes of the C57BL/6J and 129/SV mouse strains, and to study and explore the impact of the nucleotide difference on the processing of the Aanat transcript.
Materials and Methods
Animals & Tissue Collection
Four month-old wild type 129/SV and wild type C57BL/6J Mus musculus males were sacrificed one hour after light onset. Eyes were removed and the retina and retinal pigment epithelium were isolated. The pineal gland and frontal cortex were harvested.
DNA Purification & PCR
DNA from remaining brain tissues of the 129/SV and C57BL/6J were isolated and purified. Primers were designed such that they flanked the nucleotide of interest. PCR was performed, and products were purified and sequenced.
RNA Purification & RT-PCR
Frozen tissues were placed in 350µL of lysis buffer and homogenized using the tissue rupture. RNA was isolated and purified from pineal gland and frontal cortex.
RT-PCR was performed. Primers were designed (1) in exons three and four, flanking the SNP of interest and (2) in exon three and specifically including the pseudo-exon. Products generated were purified and sequenced to confirm product identity.
PCR & Genomic Sequencing
Aanat DNA sequence alignments comparing 129/SV and C57BL/6J. As expected, in intron three of the 129/SV genomic sequence, there is a guanine instead of an adenine as indicated by the green highlight (Fig. 2). The presence of an adenine nucleotide in the intronic sequence of C57BL/6J introduces a potential splice acceptor site.
RT-PCR was performed on RNA isolated from pineal gland and frontal cortex (FC) in 129/SV and C57BL/6J strains, using primers between exons three and four of the Aanat transcript. Upon visualization and sequencing, it appears that the pineal gland and FC of the C57BL/6J both show an inclusion of an additional 102bp as seen in the higher bands of C57BL/6J samples relative to that of the 129/SV samples (Fig. 3A).
In a subsequent RT-PCR, using the same RNA samples previously mentioned, primers were designed in exon three and specifically within the pseudo-exon. Products are only expressed in the C57BL/6J samples and not in the 129/SV samples (Fig. 3B). After sequence analysis, this reveals that the presence of the pseudo-exon in the Aanat transcript is only found in the C57BL/6J strain.
Comparison of Sequence Alignments
Aanat DNA sequence alignments for 129/SV and C57BL/6J compared to a reported genomic sequence of C57BL/6J (R_C57) (Fig. 4). As reported in the third intronic sequence of the C57BL/6J, the presence of the adenine instead of a guanine is being utilized as a splice acceptor site. Analysis of this reveals the insertion of a pseudo-exon via the new acceptor (bolded nucleotides) and donor (underlined nucleotides) sites, used in the C57BL/6J strain and not in the 129/SV.
The different genomic sequences of the 129/SV versus the C57BL/6J indicates a SNP between these two mouse strains (Fig. 2). The guanine-to-adenine base change in the C57BL/6J strain may not seem significant; however, since this mutation occurs adjacent to a guanine, this promotes the creation of a new splice acceptor site within the third intron (Fig. 3A, B) (Roseboom 1998). Having a new acceptor site subsequently generates a new donor site (102bp downstream the guanine-to-adenine base change) to interact with the splice acceptor site at the fourth exon in the genomic sequence (Fig. 4). These new splicing sites result in the inclusion of a pseudo-exon, which contains a premature stop codon. Having this creates a truncated Aanat protein with <1% enzymatic activity.
Future work would include further confirming the melatonin deficiency from a chemical perspective in the C57BL/6J versus the 129/SV strains by performing a melatonin ELISA. So far, a standard curve for this competitive assay has been established, but more controlled data collection will be taken place. Having this crucial piece of evidence would allow for subsequent analysis of melatonin and its effects on the circadian photoneuroendocrine system, as well as photoreceptor outer-segment renewal, by analyzing genes such as Clock, Bmal1, and Per in retinal tissues (Sakamoto and Ishida 1998).
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