Abstract
Recursive splicing (RS) is a splicing mechanism to remove long introns from messenger RNA precursors of long genes. Compared to the hundreds of RS events identified in humans and drosophila, only ten RS events have been reported in mice. To further investigate RS in mice, we analyzed RS in the mouse brain, a tissue that is enriched in the expression of long genes. We found that nuclear total RNA sequencing is an efficient approach to investigate RS events. We analyzed 1.15 billion uniquely mapped reads from the nuclear total RNA sequencing data in the mouse cerebral cortex. Unexpectedly, we only identified 20 RS sites, suggesting that RS is a rare event in the mouse brain. We also identified that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. In addition, we found that the primary sequence context is associated with RS splicing intermediates and distinguishes RS AGGT site from non-RS AGGT sites, indicating the importance of the primary sequence context in RS sites. Moreover, we discovered that cryptic exons may use an RS-like mechanism for splicing. Overall, we provide novel findings about RS in long genes in the mouse brain.
Highlights
Removing introns from the messenger RNA precursors is an essential step of gene expression
We found that 77% of the uniquely mapped reads from the nuclear total RNA-seq data are localized in introns, which is significantly higher than the 41% from the whole-cell total RNA-seq data and the 23% from the messenger RNA (mRNA)-seq data (P < 2.2−16, one-tailed Fisher’s Exact Test) (Fig 1C and S1A Fig)
The finding that only 19 Recursive splicing (RS) sites were identified from 506 million uniquely mapped reads indicates that RS is a rare event in the mouse brain
Summary
Removing introns from the messenger RNA (mRNA) precursors is an essential step of gene expression. This step is affected by the intron lengths. A long intron represents a large RNA molecule to be removed from mRNA precursors. This large molecule poses a challenge for canonical splicing mechanism, which results in a high rate of splicing errors in long introns [1]. Recursive splicing (RS) is a splicing mechanism that removes a long intron into several smaller segments as opposed to in a large single unit [2,3,4,5,6,7,8,9,10,11,12]. Whole-cell ribosomal RNA-depleted total RNA sequencing (total RNA-seq) and nascent RNA-seq have been used to identify RS splicing intermediates [4, 6, 8,9,10, 12]
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