Ndt80 activates the meiotic ORC1 transcript isoform and SMA2 via a bi-directional middle sporulation element in Saccharomyces cerevisiae

Abstract

The origin of replication complex subunit ORC1 is important for DNA replication. The gene is known to encode a meiotic transcript isoform (mORC1) with an extended 5 0 -untranslated region (5 0 -UTR), which was predicted to inhibit protein translation. However, the regulatory mechanism that controls the mORC1 transcript isoform is unknown and no molecular biological evidence for a role of mORC1 in negatively regulating Orc1 protein during gametogenesis is available. By interpreting RNA profiling data obtained with growing and sporulating diploid cells, mitotic haploid cells, and a starving diploid control strain, we determined that mORC1 is a middle meiotic transcript isoform. Regulatory motif predictions and genetic experiments reveal that the activator Ndt80 and its middle sporulation element (MSE) target motif are required for the full induction of mORC1 and the divergently transcribed meiotic SMA2 locus. Furthermore, we find that the MSE-binding negative regulator Sum1 represses both mORC1 and SMA2 during mitotic growth. Finally, we demonstrate that an MSE deletion strain, which cannot induce mORC1, contains abnormally high Orc1 levels during post-meiotic stages of gametogenesis. Our results reveal the regulatory mechanism that controls mORC1, highlighting a novel developmental stage-specific role for the MSE element in bi-directional mORC1/SMA2 gene activation, and correlating mORC1 induction with declining Orc1 protein levels. Because eukaryotic genes frequently encode multiple transcripts possessing 5 0 -UTRs of variable length, our results are likely relevant for gene expression during development and disease in higher eukaryotes.

Transcription of a 5’ extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter

“…During budding yeast meiosis, more than 190 genes express an extended 5’ leader sequence ( Brar et al, 2012 ). Some examples have been further confirmed and show a clear inhibitory effect on expression from the downstream promoter ( Liu et al, 2015 ; Xie et al, 2016 ). However, how the expression of different mRNA isoforms regulate gene expression remains unexplored at the genome-wide level.…”

Kinetochore inactivation by expression of a repressive mRNA

“…Analysis of the mRNA-seq and ribosome profiling datasets of meiotic yeast revealed hundreds of transcripts with potential luti-like signatures ( Brar et al, 2012 ). In support of this idea, two other genes, ORC1 and BOI1 , have been shown to express meiosis-specific transcript isoforms with uORF-containing leader extensions ( Xie et al, 2016 and Liu et al, 2015 ). Rather than dissecting each candidate luti-mRNA on a case by case basis, future studies that integrate additional genome-wide datasets to measure stage-specific transcription factor binding sites, transcription-coupled chromatin modification states, mRNA translation status with isoform specificity and protein abundance would result in a high-confidence map of luti-mRNAs and aid in the dissection of their cellular functions.…”

Pervasive, Coordinated Protein-Level Changes Driven by Transcript Isoform Switching during Meiosis

“…Both genes have recently been shown to be associated with regulation involving mutually exclusive alternate transcript isoforms, so we investigated the possibility that this could account for their poor mRNA:protein agreement. A recent study showed that the 5′ extended ORC1 transcript isoform results from Ndt80 activation of an upstream Transcription Start Site (TSS), producing a transcript that is poorly translated for the ORC1 ORF and instead shows translation of several upstream Open Reading Frames [uORFs;(Brar et al, 2012; Xie et al, 2016)]. Comparison to a canonical Ndt80 target, CDC5 (Fig.…”