Kinetochore inactivation by expression of a repressive mRNA
- University of California, Berkeley, United States
- The Francis Crick Institute, United Kingdom
- Li Ka Shing Center, University of California, United States
- University of California, United States
Abstract
Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5' leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.
Data availability
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High-resolution view of the yeast meiotic program revealed by ribosome profilingPublicly available at the NCBI Gene Expression Omnibus (accession no. GSE34082).
Article and author information
Author details
Folkert Jacobus van Werven
Funding
March of Dimes Foundation (5-FY15-99)
- Elcin Unal
Pew Charitable Trusts (27344)
- Elcin Unal
Glenn Foundation for Medical Research
- Elcin Unal
Francis Crick Institute
- Folkert Jacobus van Werven
National Science Foundation
- Jingxun Chen
Agency for Science, Technology and Research
- Minghao Chia
Damon Runyon Cancer Research Foundation (35-15)
- Elcin Unal
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Chen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Kinetochore inactivation by expression of a repressive mRNA
eLife 6:e27417.
https://doi.org/10.7554/eLife.27417
Further reading
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- Chromosomes and Gene Expression
A key protein involved in the segregation of meiotic chromosomes is produced 'just in time' by the regulated expression of two mRNA isoforms.
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- Chromosomes and Gene Expression
- Genetics and Genomics
Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.
