Sleep and diurnal alternative polyadenylation sites associated with human APA-linked brain disorders

Abstract

Disruption of sleep and circadian rhythms are a comorbid feature of many pathologies, and can negatively influence many health conditions, including neurodegenerative disease, metabolic illness, cancer, and various neurological disorders. Genetic association studies linking sleep and circadian disturbances with disease susceptibility have mainly focused on changes in gene expression due to mutations, such as single-nucleotide polymorphisms. The interaction between sleep and/or circadian rhythms with the use of Alternative Polyadenylation (APA) has been largely undescribed, particularly in the context of other disorders. APA generates transcript isoforms by utilizing various polyadenylation sites (PASs) from the same gene affecting its mRNA translation, stability, localization, and subsequent function. Here we identified unique APAs expressed in rat brain over time-of-day, immediately following sleep deprivation, and the subsequent recovery period. From these data, we performed a secondary analysis of these sleep- or time-of-day associated PASs with recently described APA-linked human brain disorder susceptibility genes.

Keywords: Circadian rhythms and sleep; Diseases.

Fig. 1. Schema of the brain region sampled, the collection time/condition, and a plot of the number of APA sites per gene.

A The region of the central rat forebrain that was collected and used for RNA extraction is bounded by dotted lines and labeled ‘forebrain’ (brain illustration was generated by modifying an image obtained in Motifolio (Motifolio Inc., Ellicott City, MD, USA). B For sleep homeostasis experiments, rats were sleep-deprived for 6 h and allowed to recover for 0–8 h before tissue extraction. Three of the time-matched controls (no SD) were shared with the diurnal experiment and one additional time point (no SD at ZT8), was not in common. For the diurnal analysis, samples were taken at 4 h intervals from ZT2 until ZT22. Five biological replicates were used for all data points. C A diagram of a generic gene shows different types of APAs: within an internal exon (1); within an early intron (2); following an internal exon (3); within the longest documented 3′ UTR (4); at the terminus of the longest documented 3′ UTR (5); and distal to longest documented 3′ UTR. D WTTS-seq PAS results; the number of genes on the x-axis (log10 scale) are plotted against the number of APA sites per gene.

Fig. 2. Gene ontology and pathway analysis of genes with diurnal APA site expression.

The top 10 gene ontology terms and pathways identified by WebGestalt using the 1173 genes with APAs that exhibited time-of-day oscillations and had 2 or more total APAs. Minus log10 FDRs are plotted for each GO and pathway description.

Fig. 3. Differential expression of PASs by DESeq-2 with Apeglm Shrinkage.

Log of adjusted p-values are plotted against log2 fold changes from (A) ZT6 vs R0 and (B) ZT10 vs R4.

Fig. 4. The Sin3b gene and 3′ UTR regions of the Mapt and Ntrk2 genes.

A A map of the entire rat Sin3b gene depicts exons, introns and short and long APA sites (S and L). The corresponding genes in mouse and human are extremely similar. B The average normalized read counts ±SE (y-axis) of the short (diurnal) and long Sin3b APAs are plotted against time-of-day (x-axis). C Maps of the 3′ UTR regions of the human, mouse, and rat Mapt genes are shown. Arrows labeled 1–5 indicate the positions of APA sites found in rat. The human and mouse genes have 2 APAs depicted by red and black arrows respectively. In human MAPT, APA usage correlates with several brain disorders. RNA-seq coverage from individuals homozygous for the less common SNP allele that is associated with longer transcripts (adapted from Cui. et al. ) is shown above the human MAPT 3′ UTR map. Binding sites for TDP-43 (indicated by red arrows) that were experimentally determined in mouse align with putative sites in the rat gene, and one possible TDP-43 binding site is indicated in the human 3′ UTR. The significantly diurnal APA is marked with an asterisk. Blocks of homologous sequence between the rat and human genes that were found by BLAST search are indicated by purple bars. Conserved predicted mir-181-5p binding sites are marked by red bars near the 3′ end in all three species, while two of the four predicted human mir-150-5p binding sites align with two in the rodent genes (black bars). The 3′ UTR lengths are 4380, 4119, and 3946 n.t. for human, mouse, and rat, respectively. D The average normalized read counts ±SE (y-axis) of the short Mapt isoforms lacking TDP binding sites (1 + 2) and the sum of the three longer isoforms (3 + 4 + 5) plotted against time-of-day (x-axis) are shown. E The 3′ UTR of tyrosine kinase-deficient (TK-) isoforms of the human, mouse, and rat Ntrk2 TK- genes are shown. Arrows indicate the positions of APA sites. The depicted rat APAs are from this current dataset. Diurnal rat APAs are indicated with asterisks. The 3′ UTR lengths are 5125, 5008, and 8004 n.t. for human, mouse, and rat, respectively. Mouse and rat sequence comparison by BLAST produced 4 segments having 91%, 83%, 86% and 82% identity for regions 1, 2, 3 and 4, depicted by blue bars.

Fig. 5. Map and APA read analyses of Sorl1.

A Maps of the human, mouse and rat Sorl1 gene 3′ UTRs show APA sites indicated by arrows. Four highly conserved miR binding sites are marked by red bars in all three species. The first 2 are recognized by multiple miRs. The size of dark blue bars under the rat APAs depict the individual proportion compared to the total of all WTTS Sorl1 reads. The human APAs are from established isoforms which also include different exon configurations. The first 4 mouse APAs are suggested by ESTs, and, in the latter 3 cases, by upstream polyA signals and PolyA_DB v3 data. Red ‘c’s indicate matches to the consensus CPE sites. B The proportion each Sorl1 APA contributes to the total for the gene are plotted for each of the diurnal timepoints. C The proportion each Sorl1 APA contributes to the total for the gene is plotted for the differentially expressed samples: ZT10 and 4 h after SD. D Graph of normalized read numbers of 4 Sorl1 APAs that either cycle with 24 h (M4 and L6) or 12 h (L7) hours and the one differentially expressed after SD (S1). Total reads and the corresponding scale are in red.