DOI: 10.7272/q6t72fp1
title: Data from: Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom

description: Chapter 1: Eukaryotic protein synthesis generally initiates at a start
 codon defined by an AUG and its surrounding Kozak sequence context, but
 the quantitative importance of this context in different species is
 unclear. We tested this concept in two pathogenic Cryptococcus yeast
 species by genome-wide mapping of translation and of mRNA 5’ and 3’ ends.
 We observed thousands of AUG-initiated upstream open reading frames
 (uORFs) that are a major contributor to translation repression. uORF use
 depends on the Kozak sequence context of its start codon, and uORFs with
 strong contexts promote nonsense-mediated mRNA decay. Transcript leaders
 in Cryptococcus and other fungi are substantially longer and more
 AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode
 predicted dual-localized proteins, including many aminoacyl-tRNA
 synthetases, in which a leaky AUG start codon is followed by a strong
 Kozak context in-frame AUG, separated by mitochondrial-targeting sequence.
 Analysis of other fungal species shows that such dual-localization is also
 predicted to be common in the ascomycete mould, Neurospora crassa.
 Kozak-controlled regulation is correlated with insertions in translational
 initiation factors in fidelity-determining regions that contact the
 initiator tRNA. Thus, start codon context is a signal that quantitatively
 programs both the expression and the structures of proteins in diverse
 fungi. Chapter 2: The human pathogenic yeast Cryptococcus neoformans
 silences transposable elements using endo-siRNAs and an Argonaute, Ago1.
 Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1,
 and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is
 independent of histone H3 lysine 9 methylation. We describe here an
 insertional mutagenesis screen for factors required to suppress the
 mobilization of the C. neoformans HARBINGER family DNA transposon HAR1.
 Validation experiments uncovered five novel genes (RDE1-5) required for
 HAR1 suppression and global production of suppressive endo-siRNAs. The RDE
 genes do not impact transcript levels, suggesting the endo-siRNAs do not
 act by impacting target transcript synthesis or turnover.  RDE3
 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes
 a predicted terminal nucleotidyltransferase, while RDE5 has no strongly
 predicted encoded domains.  Affinity purification-mass
 spectrometry studies suggest that Rde3 and Rde5 are physically associated.
 RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1,
 a nucleolar protein that directly activates the essential helicase Prp43
 during rRNA biogenesis.  Rde1 copurifies Rde2, another novel
 protein obtained in the screen, as well as Ago1, a homolog of Prp43, and
 numerous predicted nucleolar proteins. We also describe the isolation of
 conditional alleles of PRP43, which are defective in RNAi. This work
 reveals unanticipated requirements for a non-Dicer RNase III and
 presumptive nucleolar factors for endo-siRNA biogenesis and transposon
 mobilization suppression in C. neoformans. Chapter 3: Tools to understand
 how the spliceosome functions in vivo have lagged behind advances in its
 structural biology. We describe methods to globally profile
 spliceosome-bound precursor, intermediates and products at nucleotide
 resolution. We apply these tools to three divergent yeast species that
 span 600 million years of evolution. The sensitivity of the approach
 enables detection of novel cases of non-canonical catalysis including
 interrupted, recursive and nested splicing. Employing statistical modeling
 to understand the quantitative relationships between RNA features and the
 data, we uncover independent roles for intron size, position and number in
 substrate progression through the two catalytic stages. These include
 species-specific inputs suggestive of spliceosome-transcriptome
 coevolution. Further investigations reveal ATP-dependent discard of
 numerous endogenous substrates at both the precursor and
 lariat-intermediate stages and connect discard to intron retention, a form
 of splicing regulation. Spliceosome profiling is a quantitative,
 generalizable global technology to investigate an RNP central to
 eukaryotic gene expression. Chapter 4: We determined that over 60
 spliceosomal proteins are conserved between many fungal species and humans
 but were lost during the evolution of S. cerevisiae, an
 intron-poor yeast with unusually rigid splicing
 signals.  We analyzed null mutations in a subset of
 these factors, most of which had not been investigated previously, in the
 intron-rich yeast Cryptococcus neoformans.  We
 found they govern splicing efficiency of introns with divergent spacing
 between intron elements.  Importantly, most of these
 factors also suppress usage of weak nearby cryptic/alternative splice
 sites.  Among these, orthologs of GPATCH1 and the
 helicase DHX35 display correlated functional signatures and copurify with
 each other as well as components of catalytically active spliceosomes,
 identifying a conserved G-patch/helicase pair that promotes splicing
 fidelity.  We propose that a significant fraction of
 spliceosomal proteins in humans and most eukaryotes are involved in
 limiting splicing errors, potentially through kinetic proofreading
 mechanisms, thereby enabling greater intron diversity.