Post-transcriptional regulation of spermatogenesis through intron retention in the fern Marsilea vestita

Abstract

Many rapidly developing systems rely on the use of stored transcripts to carry out their developmental program. The microspore of M. vestita transcribes and stores RNA during a requisite period of desiccation. Rehydration of the microspore triggers spermatogenesis to begin, a process that is mediated by the utilization of these stored RNAs. Here I investigate mechanisms controlling the spatial and temporal utilization of these stored transcripts. Next generation Solexa based RNAseq was conducted using poly(A)+ RNA isolates from specific time ranges during spermatogenesis. A reference transcriptome as well as temporally specific transcriptomes were assembled de novo and analyzed for gene ontology enrichments. This analysis revealed an overrepresentation of catalytic splicing and nuclear speckle factors early in development suggesting that some transcripts are not fully mature. An in house Visual Basic for Applications program was used to identify potential intron retaining transcripts (IRTs) within our transcriptomes. A large subset of IRTs was identified and in silico and molecular biological approaches demonstrated that these IRTs are matured in a spliceosome dependent fashion at different times during development. Intron retention appears to confer a translational block to IRTs and splicing of retained introns alleviates this block. IRTs appear to be associated with splicing machinery organized in nuclear speckles. These subnuclear domains aggregate during desiccation and upon rehydration are proportioned asymmetrically to spermatogeneous cells. It appears that intron retention mediates both the association and asymmetric distribution of IRTs with nuclear speckles as well as their temporal utilization through post-transcriptional splicing.