Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom

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.

Chapter 1:
真核生物蛋白质合成通常始于由AUG起始密码子及其侧翼科扎克序列背景（Kozak sequence context）所界定的起始位点，但该背景在不同物种中的定量重要性仍不明晰。我们通过全基因组范围的翻译图谱分析以及mRNA 5'端和3'端定位，在两种致病隐球菌（Cryptococcus）酵母物种中验证了这一观点。我们观测到数千个由AUG起始的上游开放阅读框（upstream open reading frames, uORFs），它们是翻译抑制的主要贡献因素之一。上游开放阅读框的使用情况取决于其起始密码子的科扎克序列背景，而具备强科扎克背景的上游开放阅读框会促进无义介导的mRNA降解（nonsense-mediated mRNA decay）。隐球菌与其他真菌的转录前导序列相较于酿酒酵母（Saccharomyces）显著更长，且AUG位点密度更高。诸多隐球菌mRNA编码预测的双定位蛋白，其中包含众多氨酰-tRNA合成酶（aminoacyl-tRNA synthetases）：这类蛋白的起始处存在一个漏扫型AUG起始密码子，其后紧跟着一个具备强科扎克背景的框内AUG，二者由线粒体靶向序列分隔。对其他真菌物种的分析显示，这类双定位现象在子囊菌霉菌粗糙脉孢霉（Neurospora crassa）中也被预测为普遍存在。受科扎克序列调控的机制与翻译起始因子中与起始tRNA结合的保真度决定区域内的插入序列相关。由此可见，起始密码子背景是一种可定量调控多种真菌中蛋白质表达与结构的信号。

Chapter 2:
致病人类酵母新型隐球菌（Cryptococcus neoformans）通过内源小干扰RNA（endo-siRNAs）与Argonaute蛋白Ago1来沉默转座因子。内源小干扰RNA的生成依赖于RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase）Rdp1以及两种部分功能冗余的Dicer酶Dcr1和Dcr2，但不依赖于组蛋白H3赖氨酸9甲基化（histone H3 lysine 9 methylation）。本研究针对新型隐球菌HARBINGER家族DNA转座子HAR1的转座抑制所需因子开展了插入诱变筛选（insertional mutagenesis screen）。验证实验揭示了5个参与HAR1抑制以及全局抑制性内源小干扰RNA生成的新基因（RDE1-5）。RDE基因并不影响转录本水平，这表明内源小干扰RNA并非通过影响靶标转录本的合成或降解来发挥作用。RDE3编码一种与酿酒酵母Rnt1同源的非Dicer型RNase III蛋白；RDE4编码一个预测的末端核苷酸转移酶（terminal nucleotidyltransferase）；而RDE5则未被预测到存在明确的编码结构域。亲和纯化-质谱联用（affinity purification-mass spectrometry）实验结果显示Rde3与Rde5存在物理相互作用。RDE1编码一个G-patch结构域蛋白，与酿酒酵母Sqs1/Pfa1同源——后者是一种在核糖体RNA（rRNA）生物发生过程中直接激活必需解旋酶Prp43的核仁蛋白。Rde1可与筛选中获得的另一新蛋白Rde2、Prp43的同源蛋白Ago1以及众多预测的核仁蛋白共纯化。本研究还报道了PRP43的条件性等位基因的分离，这类等位基因在RNA干扰（RNAi）过程中存在功能缺陷。本研究揭示了新型隐球菌内源小干扰RNA生成以及转座子转座抑制过程中，此前未被认知的非Dicer型RNase III以及推定的核仁因子需求。

Chapter 3:
解析剪接体（spliceosome）在活体内功能的工具相较于其结构生物学研究进展始终滞后。我们开发了可在核苷酸分辨率（nucleotide resolution）下全局分析与剪接体结合的前体RNA、中间产物与最终产物的方法。我们将这些方法应用于三个跨越6亿年进化历程的不同酵母物种。该方法的高灵敏度可帮助我们检测到非经典催化（non-canonical catalysis）的新型案例，包括间断式剪接、递归式剪接与嵌套式剪接。我们通过统计建模解析RNA特征与实验数据间的定量关系，发现内含子的长度、位置与数量在底物经历两个催化阶段的过程中各自发挥独立作用。其中包含物种特异性的调控机制，这暗示了剪接体与转录组的协同进化。进一步研究显示，在剪接前体与套索中间产物阶段，大量内源底物会以ATP依赖的方式被丢弃，并将该丢弃过程与内含子保留（intron retention），一种剪接调控形式，相关联。剪接体图谱分析是一种可量化、可推广的全局技术，用于研究真核基因表达的核心核糖核蛋白复合物（RNP）。

Chapter 4:
我们发现，在众多真菌物种与人类之间保守存在的剪接体蛋白多达60余种，但这些蛋白在酿酒酵母的进化过程中丢失了——酿酒酵母是一种内含子稀少且剪接信号异常严格的酵母。我们在富含内含子的新型隐球菌中分析了这类因子中一部分的敲除突变体（null mutations），其中大多数因子此前未被研究过。我们发现这些因子可调控内含子元件间距存在差异的内含子的剪接效率。重要的是，这类因子中的大多数还会抑制邻近的弱识别隐蔽剪接位点（cryptic splice sites）/可变剪接位点（alternative splice sites）的使用。其中，GPATCH1的同源蛋白与解旋酶DHX35的同源蛋白展现出相关的功能特征，且二者可彼此共纯化，同时还可与具有催化活性的剪接体组分结合，由此确定了一对保守的G-patch/解旋酶蛋白对，其可提升剪接保真度。我们提出，人类与大多数真核生物中，有相当一部分剪接体蛋白参与限制剪接错误，其机制可能涉及动力学校正（kinetic proofreading mechanisms），从而实现更多样的内含子类型。