Analysis of subcellular RNA fractions demonstrates significant genetic regulation of gene expression in human brain post-transcriptionally

Abstract

Gaining insight into the genetic regulation of gene expression in human brain is key to the interpretation of genome-wide association studies for major neurological and neuropsychiatric diseases. Expression quantitative trait loci (eQTL) analyses have largely been used to achieve this, providing valuable insights into the genetic regulation of steady-state RNA in human brain, but not distinguishing between molecular processes regulating transcription and stability. RNA quantification within cellular fractions can disentangle these processes in cell types and tissues which are challenging to model in vitro. We investigated the underlying molecular processes driving the genetic regulation of gene expression specific to a cellular fraction using allele-specific expression (ASE). Applying ASE analysis to genomic and transcriptomic data from paired nuclear and cytoplasmic fractions of anterior prefrontal cortex, cerebellar cortex and putamen tissues from 4 post-mortem neuropathologically-confirmed control human brains, we demonstrate that a significant proportion of genetic regulation of gene expression occurs post-transcriptionally in the cytoplasm, with genes undergoing this form of regulation more likely to be synaptic. These findings have implications for understanding the structure of gene expression regulation in human brain, and importantly the interpretation of rapidly growing single-nucleus brain RNA-sequencing and eQTL datasets, where cytoplasm-specific regulatory events could be missed.

Figure 1

(a) Experimental Overview: The cerebellar cortex (4 individuals), putamen (3 individuals) and anterior prefrontal cortex (4 individuals) regions of the brain, were sampled from human control brains. Separate cellular fractions for each tissue sampled were obtained by centrifugation and RNA extracted using spin column chromatography resulting in a total of 22 nuclear and cytoplasmic samples. (b) Plot showing the quality of fractionation assessed by examining the enrichment of ACTB in the cytoplasmic and MALAT1 in the nuclear fraction. A positive log₂foldchange indicates gene expression in the nucleus is higher while a negative value indicates the gene expression in the cytoplasm is higher.

Figure 2

Volcano plots showing the genes with at least double expression (log₂foldchange >|1|) per tissue. Positive values = expression in the nuclear fraction is higher and negative values = higher expression in the cytoplasmic fraction. 2 outlier datapoints (p values 7.94E-41 and 5.69E-55) have been excluded from the putamen lncRNA plot. Note: The y-axis scales differ between the plots.

Figure 3

Visualising tissue differences. Plots showing the paired $\left|a,l,l,e,l,i,c,r,a,t,i,o,-,0.5\right|$ (where allelic ratio is defined as the proportion of reference allelic counts) values for each hetSNP with ASE signals in both fractions, cytoplasm-specific and nucleus-specific. Each point represents a hetSNP in an individual and tissue. The x-axis represents the $\left|a,l,l,e,l,i,c,r,a,t,i,o,-,0.5\right|$ value for the hetSNP in the nuclear fraction and y-axis its corresponding value in the cytoplasmic fraction. Red line = regression line.