RNGTT

Through this partnership, Sensible and Ginkgo aim to develop an in vivo alternative to conventional in vitro mRNA production technology OXFORD, United Kingdom, April 3, 2023 /PRNewswire/ -- Sensible Biotechnologies, an early stage biotechnology company, today announced a partnership to develop an in vivo microbial mRNA manufacturing platform with Ginkgo Bioworks (NYSE: DNA), which is building the leading platform for cell programming and biosecurity. Today, the vast majority of mRNA used in vaccines, therapeutics and other applications is produced by in vitro transcription (IVT), a cell-free process, which is driven by purified enzymes. In conventional mRNA manufacturing, production runs are typically limited in reaction volume size, and the resulting mRNA needs to go through expensive purification processes to eliminate potentially harmful byproducts like dsRNA, which can cause adverse immune responses in patients. Moreover, it is difficult to produce high yields of certain kinds of mRNA in an IVT reaction. By contrast, the in vivo mRNA manufacturing method Sensible and Ginkgo are working to develop is designed to scale to upwards of 100,000L, with the goal of superior quality mRNA over traditional IVT, allowing for the production of mRNA molecules with increased length and expanding the potential of the mRNA platform to novel therapeutic modalities. "mRNA technology has a potential to bring many life-saving therapeutics and vaccines, but its current, cell-free production represents one of the major bottlenecks. In vivo mRNA manufacturing could enable scalable mRNA manufacturing, which has long relied on production methods that face quality control challenges and are inherently difficult to scale," said Miroslav Gasparek, CEO at Sensible. "By working with Ginkgo, we aim to create a scalable commercial-grade manufacturing platform that produces mRNA of higher quality than is possible through in vitro expression and enable the advent of novel mRNA medicines." "As the market for mRNA continues to expand, biopharma companies are looking for more efficient and scalable production platforms to produce high-quality mRNA," said Austin Che, co-founder and Head of Strategy at Ginkgo Bioworks. "As Ginkgo deepens its commitment to the emerging field of nucleic acid therapeutics, we are excited to work with the team at Sensible Biotechnologies to optimize their microbial mRNA production platform with the goal of unleashing a new generation of mRNA products." This partnership with Ginkgo builds on Ginkgo's existing pipeline of mRNA-related programs. Ginkgo worked with Moderna to support process optimization for raw materials used to make mRNA vaccines. Furthermore, through its partnership with Aldevron, Ginkgo optimized the production of vaccinia capping enzyme, an important component often required to manufacture mRNA vaccines and therapeutics. Most recently, Ginkgo announced the acquisition of Circularis, a biotechnology company with a proprietary circular RNA and promoter screening platform, and is actively engaged in improving circular RNA efficacy and manufacturing yields. About Sensible Sensible Biotechnologies is building a cell-based platform for the production of high-quality mRNA. Sensible's platform eliminates the major bottlenecks in the mRNA production process in an effort to unlock the full potential of mRNA technology and the next generation of mRNA therapeutics and vaccines. Sensible aims to become an integral part of the global biomanufacturing infrastructure and biosecurity framework. Sensible Biotechnologies is based in Oxford, United Kingdom, and in Bratislava, Slovakia. For further information, visit , and follow us on Twitter @sensible_bio. About Ginkgo Bioworks Ginkgo Bioworks is the leading horizontal platform for cell programming, providing flexible, end-to-end services that solve challenges for organizations across diverse markets, from food and agriculture to pharmaceuticals to industrial and specialty chemicals. Ginkgo's biosecurity and public health unit, Concentric by Ginkgo, is building global infrastructure for biosecurity to empower governments, communities, and public health leaders to prevent, detect and respond to a wide variety of biological threats. For more information, visit ginkgobioworks.com and concentricbyginkgo.com, read our blog, or follow us on social media channels such as Twitter (@Ginkgo and @ConcentricByGBW), Instagram (@GinkgoBioworks and @ConcentricByGinkgo), or LinkedIn. SENSIBLE MEDIA CONTACT: [email protected] SOURCE Sensible Biotechnologies

Enzyme Intelligence Virtual Event on December 15, 2022 gives overview of Ginkgo's approach to machine learning-guided enzyme engineering BOSTON, Dec. 8, 2022 /PRNewswire/ -- Ginkgo Bioworks (NYSE: DNA), which is building the leading platform for cell programming and biosecurity, today announced the launch of Ginkgo Enzyme Services. Ginkgo Enzyme Services is powered by ultra high throughput screening and machine learning-guided protein design, as well as optimized proprietary bacterial and fungal host strains. Ginkgo Enzyme Services solves challenges for R&D teams developing enzymes, from discovery of novel enzyme activity through optimization of enzyme function and large scale manufacturing. A virtual event on Dec. 15 will give an overview of Ginkgo's Enzyme Intelligence approach to machine learning-guided enzyme engineering, a core element of this offering. Enzymes are valuable biocatalysts used across a wide range of industries including in the production of medicines, food, materials, and beyond. Ginkgo's end-to-end Enzyme Services support R&D leaders looking to identify new enzyme activity to replace existing chemical synthesis steps, improve enzyme specificity, activity, and stability in industrially relevant conditions, and optimize the manufacturing of enzymes for reduced cost of goods and environmental impact. Ginkgo has supported enzyme R&D programs across a wide range of industries, including biopharma manufacturing and discovery. Notable enzyme services projects include breakthrough work with Aldevron to improve the manufacturing efficiency of vaccinia capping enzyme, a critical reagent used in the production of mRNA vaccines, and a recently announced partnership with Merck to develop Biocatalysts for active pharmaceutical ingredient (API) manufacturing. Ginkgo's suite of services covers the full end-to-end process for enzyme R&D, providing synergies between enzyme sequence, host strain, and manufacturing processes that can enable commercial success. Ginkgo Enzyme Services includes: Novel Enzyme Discovery Discover novel enzyme activity and identify starting points for further protein engineering by drawing from Ginkgo's incredibly broad database of proprietary and public genomic sequences Mine genome databases using Ginkgo's in-house computational tools to identify thousands of potential hits Leverage metagenomic screens to design around existing IP that would otherwise block your development Screen candidates using advanced ultra high throughput screening methods to identify unique enzymes with the desired activity Enzyme Function & Stability Optimization Use a broad and constantly expanding toolkit of evolutionary, computational, and rational protein engineering approaches to iteratively improve catalytic activity, enzyme stability, specificity, and expression Ultra high throughput assay data empowers supervised machine learning to dramatically improve enzyme function Access to Optimized Host Strains for Robust Expression Express enzymes in Ginkgo's proprietary bacterial and fungal hosts optimized for protein production, including next-generation methanol-free Pichia pastoris and low viscosity Aspergillus niger Leverage Ginkgo's deep strain engineering expertise to optimize enzyme expression in Ginkgo's high expression host strains, tailored to support optimal expression of the enzyme to reduce production costs Optimized Fermentation Process Development Automated design of experiments at lab scale using Ginkgo's fleet of hundreds of Ambr® 250 bioreactors to quickly develop fermentation processes, with a proven record of transferring to commercial scale Manufacturing Scale-Up, Process Development & Tech Transfer Perform pilot scale fermentation in one of Ginkgo's facilities with nameplate capacities up to 3,000L Optimize downstream processing and purification methods Tech transfer to other manufacturing partners, including GMP production "Most R&D teams working on developing enzymes expect to need to stitch together a bunch of different services and tools, both in-house and external to make their enzymes work," said Jake Janey, PhD, a pioneer in the field of biocatalysis. "The ability for Ginkgo to guide the process all the way, providing many intermediate touchpoints with data and prototypes for development and decision making, is a great value-add." "At Ginkgo, we are constantly working to improve our platform to provide best-in-class services to enable our customers to meet their R&D challenges head on. As a horizontal platform, we have the flexibility, breadth, and scale to serve customers across biocatalysis, diagnostics, and beyond with the full spectrum of tools they need to discover, develop, and deploy enzymes for their processes and products," said Behzad Mahdavi, SVP of Biopharma Manufacturing & Life Sciences Tools. "With world class expression hosts, automation, computational design, and fermentation capabilities, we are excited to partner with companies across all industries to bring their enzyme-dependent products from conception to market more swiftly and reliably than ever before." Virtual event details: Join us for Enzyme Intelligence, a virtual event featuring Ginkgo's head of protein engineering, Emily Wrenbeck, on December 15, 2022. Sign up here. Learn more about Ginkgo Enzyme Services at our webpage, or write to us at enzymes@ginkgobioworks.com. About Ginkgo Bioworks Ginkgo Bioworks is the leading horizontal platform for cell programming, providing flexible, end-to-end services that solve challenges for organizations across diverse markets, from food and agriculture to pharmaceuticals to industrial and specialty chemicals. Ginkgo's biosecurity and public health unit, Concentric by Ginkgo, is building global infrastructure for biosecurity to empower governments, communities, and public health leaders to prevent, detect and respond to a wide variety of biological threats. For more information, visit ginkgobioworks.com and concentricbyginkgo.com, read our blog, or follow us on social media channels such as Twitter (@Ginkgo and @ConcentricByGBW), Instagram (@GinkgoBioworks and @ConcentricByGinkgo), or LinkedIn. Forward-Looking Statements of Ginkgo Bioworks This press release contains certain forward-looking statements within the meaning of the federal securities laws, including statements regarding the capabilities and potential success of the enzyme services and Ginkgo's cell programming platform. These forward-looking statements generally are identified by the words "believe," "can," "project," "potential," "expect," "anticipate," "estimate," "intend," "strategy," "future," "opportunity," "plan," "may," "should," "will," "would," "will be," "will continue," "will likely result," and similar expressions. Forward-looking statements are predictions, projections and other statements about future events that are based on current expectations and assumptions and, as a result, are subject to risks and uncertainties. Many factors could cause actual future events to differ materially from the forward-looking statements in this press release, including but not limited to: (i) volatility in the price of Ginkgo's securities due to a variety of factors, including changes in the competitive and highly regulated industries in which Ginkgo operates and plans to operate, variations in performance across competitors, and changes in laws and regulations affecting Ginkgo's business, (ii) the ability to implement business plans, forecasts, and other expectations, and to identify and realize additional business opportunities, (iii) the risk of downturns in demand for products using synthetic biology, (iv) the unpredictability of the duration of the COVID-19 pandemic and the demand for COVID-19 testing and the commercial viability of our COVID-19 testing business, (v) changes to the biosecurity industry, including due to advancements in technology, emerging competition and evolution in industry demands, standards and regulations, and (vi) our ability to realize the expected benefits of merger and acquisition transactions. The foregoing list of factors is not exhaustive. You should carefully consider the foregoing factors and the other risks and uncertainties described in the "Risk Factors" section of Ginkgo's quarterly report on Form 10-Q filed with the U.S. Securities and Exchange Commission (the "SEC") on November 14, 2022 and other documents filed by Ginkgo from time to time with the SEC. These filings identify and address other important risks and uncertainties that could cause actual events and results to differ materially from those contained in the forward-looking statements. Forward-looking statements speak only as of the date they are made. Readers are cautioned not to put undue reliance on forward-looking statements, and Ginkgo assumes no obligation and does not intend to update or revise these forward-looking statements, whether as a result of new information, future events, or otherwise. Ginkgo does not give any assurance that it will achieve its expectations. GINKGO BIOWORKS INVESTOR CONTACT: investors@ginkgobioworks.com GINKGO BIOWORKS MEDIA CONTACT: press@ginkgobioworks.com Company Codes: NYSE:DNA

摘要:真核生物转录的早期阶段包括两个主要事件：新合成mRNA的加帽，以及RNA聚合酶II从启动前复合物到生产性延伸状态的转变。加帽检查点模型意味着这些事件是紧密耦合的，这对于确保新合成mRNA的正确加帽是必要的。最近的发现还表明，加帽机制对转录和整个基因表达过程具有更广泛的影响。这些现象的分子基础将在本文中被讨论。1.引文真核生物mRNA的一个特征是5’端存在帽状结构。该结构由倒置的7-甲基鸟苷组成，其与新合成的转录子的第一个转录核苷酸相连，随后被帽结合蛋白复合物（CBC）结合。帽状结构的主要功能是通过保护mRNA免于5’核酸外切酶降解，以此来稳定新生的转录子。此外，帽状结构有助于聚集用于拼接、3’端加工、导出和翻译所需的因子。mRNA 加帽机制具有三种基本活性：RNA 5’三磷酸酶（RT）、鸟苷酸转移酶（GT）和RNA鸟嘌呤-N7甲基转移酶（RNMT）。在酵母中，有3种独立的酶负责这些活动。RNA 5’三磷酸酶（RT）和鸟苷酸转移酶（GT）在酿酒酵母中结合在一个功能复合物起作用，但在粟酒裂殖酵母中分别发挥作用。在后生动物中，前两种活性由相同的加帽酶（CE）进行，而甲基转移酶（RNMT）活性存在于 RNMT-RAM 复合物中，其中RNMT是催化亚基，RAM 是激活RNMT活性的活化亚基。此外，加帽机制包括帽子特异性mRNA-甲基转移酶 CMTR1和CMTR2（CMTr），以及最近描述的腺苷N6-甲基转移酶（CAPAM）。真核生物的mRNA加帽过程并不总是进行至完成。在哺乳动物中，DXO/Dom3Z和XRN家族蛋白在5’端加帽质量控制中用作监测蛋白质。特别是，脱帽外切核糖核酸酶DXO会降解加帽但未甲基化的早期mRNA。在酿酒酵母中，RNA加帽质量控制存在部分冗余机制：Rai1-Rat1和Ydr370C/Dxo1。当加帽和转录机制紧密耦合时，加帽发生。下面，我们描述了关于加帽过程和转录事件之间联系的观点。表1显示了在不同物种中参与建立这种联系的因素列表。2.加帽检查点模型RNA聚合酶II（Pol II）依赖型转录是一个由多种因素和信号调节的复杂过程，其中涉及多个检查点，以确保正确的mRNA合成和mRNA-蛋白质复合物的组装。主要检查点包括正确的mRNA加帽、剪接和3’端形成。真核生物转录发生在以下基本阶段：起始前复合物的组装、起始、延伸和终止。起始到延伸的转变进一步分为“早期延伸”和“生产性延伸”。这两个子阶段由RNA聚合酶II的暂停进行区分：在大约20-60个核苷酸转录后，RNA聚合酶II停止合成，直到特定信号出现。对hsp模型基因这一现象的早期研究表明，停顿与mRNA帽状结构的形成有关。加帽检查点模型表明RNA聚合酶II在启动子近端区域暂停，以确保在生产性延伸开始之前mRNA加帽。早期延伸的停止确保了加帽酶的聚集，这反过来又吸引了其他因素来取消早期延伸的停止。随着RNA聚合酶II穿过暂停区域，加帽逐渐发生：最近的暂停RNA大部分没有加帽，而更远端的完全加帽。在此上下文中，加帽意味着添加鸟苷，而其在转录过程中甲基化的时间点尚未确定。这就是为什么“加帽”在下文中指的是添加鸟苷的事件，并特别指出了其甲基化的情况。即使在暂停阶段，有几个因素可以确保进行适当的转变。转录起始后，DRB敏感型诱导因子（DSIF）结合RNA聚合酶II，并将负延伸因子（NELF）聚集到染色质。后者导致转录暂停，在此期间加帽机器聚集。在早期延伸过程中，RNA聚合酶II C末端结构域（Pol II CTD）在第5个丝氨酸残基处被细胞周期蛋白依赖型激酶7（Cdk7）磷酸化，其是TFIIH的一个亚基。磷酸化的RNA聚合酶II C末端结构域（Pol II CTD）和DRB敏感型诱导因子（DSIF）的Spt5亚基聚集加帽机器。加帽酶的聚集可以缓解负延伸因子（NELF）的作用，并为正转录延伸因子（P-TEFb）加载提供平台。后者使RNA聚合酶II C末端结构域（Pol II CTD）、DRB敏感型诱导因子（DSIF）和负延伸因子（NELF）的第2个丝氨酸残基磷酸化。结果，磷酸化的负延伸因子解离，暂停的RNA聚合酶II被释放到生产性延伸中（图1）。3.加帽机器的功能取决于转录mRNA加帽装置和早期伸长因子在细胞核中显示出紧密的联系。连接这些机器的核心参与者是RNA聚合酶II C末端结构域（Pol II CTD）和Spt5。事实上，带有截断CTD的RNA聚合酶II显示了加帽缺陷。RNA聚合酶II C末端结构域具有特定的磷酸化模式，这取决于转录过程的阶段，而加帽酶（CE）的聚集和功能取决于该模式。正是磷酸化的CTD将转录与加帽耦合。更准确地说，它在第5个丝氨酸残基处的磷酸化（而不是在Ser2处）激活了加帽活性。启动子上的第5个丝氨酸残基磷酸化提供了正确的早期mRNA加工。第5个丝氨酸残基磷酸化的RNA聚合酶II已被证明可与加帽机器和TFIIH激酶共纯化。最近的研究表明，Ser7磷酸化对于加帽也很重要：鸟苷酸转移酶 Ceg1与酵母中的RNA聚合酶II相关联是必要的。裂变酵母中的Ser5磷酸化RNA聚合酶II C末端结构域和Spt5与鸟苷酸转移酶 Pce1和三磷酸酶Pct1相互作用。在芽殖酵母中，RNA鸟苷酸转移酶 Ceg1与Ser5磷酸化CTD结合，并将Cet1三磷酸酶聚集到RNA聚合酶II，并且还表明Ceg1和Abd1直接独立地与磷酸CTD结合。Ser5磷酸化对于RNGTT结合及其在哺乳动物中的活性也很重要。人类RNA鸟嘌呤-N7甲基转移酶（RNMT）募集还依赖于Ser5磷酸化[46]，RNMT与加帽酶和RNA聚合酶II的伸长形式形成三元复合物。小鼠加帽酶Mce1的鸟苷酸转移酶结构域与磷酸化的CTD结合。此外，哺乳动物鸟苷酸转移酶通过与Ser5磷酸化CTD结合以变构方式激活。因此，加帽机器与Ser5磷酸化Pol II CTD的连接是一个保守的特征。然而，哺乳动物加帽酶中CTD结合位点的位置和组成与酵母加帽酶中的位置和组成不同，后者识别相同的CTD一级结构。在芽殖酵母中，所有3个加帽酶被聚集到转录基因的5’端需要Kin28。这是负责Ser5磷酸化的主要激酶（后生动物中TFIIH的Cdk7亚基的同源物）。抑制人Cdk7活性导致加帽酶聚集减少，从而减少mRNA加帽，并增加RNA聚合酶II启动子-近端暂停。正如预期的那样，在生产性延伸期间，起作用的Pol II CTD磷酸酶拮抗CE结合。因此，磷酸酶FCP1对于哺乳动物中CE与Pol II CTD的解离是必要的。然而，单独磷酸化的Pol II CTD不足以实现高效加帽。有一种CTD独立但Pol II介导的机制，与CTD依赖性过程并行工作，以确保最佳的加帽。酵母中的mRNA加帽酶需要两个界面才能与Pol II相互作用：Ser5磷酸化CTD和Rpb1的多螺旋足域。后者有助于CE与Pol II相互作用的特异性。足部结构域或其相关因子的突变导致Ser5磷酸化增加。在酿酒酵母中，Cet1和Ceg1以异四聚体Cet12Ceg12形式与Pol II相互作用。Ceg1与磷酸化的Pol II CTD之间的相互作用已得到充分研究，但Cet1也与CTD外部转录的Pol II复合物形成广泛的相互作用。Ceg1似乎是可移动的，并且在转录复合体中采用多种构象。还观察到Ceg1与Pol II亚基Rpb7的接触。因此，CTD非依赖性和CTD依赖性网络共享机制的组合在激活共转录加帽。另一个有助于加帽机器募集的转录因子是Spt5蛋白，它是早期Pol II伸长因子DSIF的亚基。Spt5携带CTD，其作用类似于Pol II CTD。Spt5和Pol IICTD对于CE的招募很重要。在裂变酵母中，三磷酸酶Pct1和GT Pce1独立地与伸长因子Spt5结合。在芽殖酵母中，Spt5有助于mRNA加帽酶 Cet1、Ceg1和Abd1的稳定募集。有实验证据表明“Spt5 CTD代码”的概念，类似于Pol II CTD的概念。根据这一概念，Spt5 CTD在结构上是灵活的，可以采用由特定细胞Spt5CT受体蛋白模板化的不同构象；此外，Spt5CTD 重复的苏氨酸磷酸化刻有一个二元开关，由不同的CTD受体读取，每个受体都有自己独特的方式。与Pol II CTD不同，P-TEFb磷酸化Spt5 CTD阻断了Spt5-Pce1相互作用。值得注意的是，CE招募的方式可能是物种特异性的。在芽殖酵母中，帽甲基转移酶Abd1直接与磷酸化的丝氨酸5 CTD Pol II相互作用，而裂变酵母中的帽甲基转移酶Pcm1在与Cdk9/Pch1（P-TEFb）的复合物中募集，Cdk9/Pch1显然将加帽装置靶向转录复合物。一个以上的一般转录因子参与适当的加帽。这是TFIIB尖端区域，这是丝氨酸5 CTD Pol II磷酸化和mRNA加帽的适当水平所必需的。在哺乳动物中，第一和第二转录的核苷酸也可以被CMTR1和CMTR2酶O-2甲基化。这种修饰在翻译启始和转录本识别为先天免疫中的“自我”方面具有作用。在转录早期将CMTR1招募到Ser5磷酸化的Pol II CTD中。最近，发现了一种新的甲基转移酶CAPAM。它催化第一个转录的腺苷N6-甲基化。CAPAM被鉴定为Ser5磷酸化CTD相互作用因子1（PCIF1）。虽然最初发现CAPAM对模型基因的RNA聚合酶II依赖性转录有负面影响[68]，但后来的全转录组分析显示其对转录有基因特异性影响。因此，CE与Pol II和Spt5之间存在许多相互作用。这些互动对于CE的招募和功能至关重要。加帽机器与转录装置之间的连接是双峰的。一方面，Pol II CTD可以作为将mRNA 5’端和CE结合在一起的支架，从而提高加帽效率；另一方面，转录因子可以变构地增强加帽机器的活性。4.加帽装置对转录的影响加帽酶（CEs）与转录机制之间的关系是相互的。加帽酶与CTD Pol II的相互作用增强了加帽，但加帽酶也会影响转录。如初始所示，Cet1抑制转录再启动，并降低RNA聚合酶II在启动子-近端区域的积累，该区域独立于mRNA加帽活性。Ceg1刺激早期伸长，其突变体在此过程中存在缺陷。最新发现表明，Cet1-Ceg1复合物和DSIF刺激Pol II启动子逃逸并转移到伸长。酵母甲基转移酶Abd1对启动子的Pol II募集具有基因特异性刺激作用。Abd1对转录的刺激发生在甲基化缺陷突变体中，因此与加帽本身无关。Abd1中的条件突变体在某些基因上与启动子结合的Pol II以及在其他基因的启动子清除或早期伸长方面存在缺陷。Abd1 耗竭导致 CTD Ser5的高磷酸化。裂变酵母加帽-甲基转移酶Pcm1将P-TEFb招募到染色质中。当这种连接被破坏时，Cdk9没有被正确招募，Pol II延伸受到严重影响。Cdk9与RNA三磷酸酶Pct1的相互作用也在粟酒裂殖酵母中进行了描述。加帽酶沿基因的分布意味着它们会影响转录的后期阶段。在酵母中，Cet1和Ceg1的全基因组占用仅限于转录起始位点，而Abd1的占用率在下游110 bp处达到峰值，CBC的占用率随后升高。据推测，Ceg1-Cet1加帽装置与转录复合物解离，因为Ser5磷酸化在延伸过程中减少，而酵母Abd1帽甲基转移酶延长了与Pol II CTD的相互作用。人类加帽酶存在于5‘末端和整个基因中，甚至包括poly（A）位点下游超过千碱基的3侧翼区域。因此，加帽因子会影响转录延伸、终止、和mRNA 3’端加工。酿酒酵母Cet1 N端结构域（NTD）促进染色质转录（FACT）的募集或促进染色质转录（FACT），从而增强在Pol II参与活性基因的转录延伸，独立与mRNA加帽活性。Cet1 N端结构域的缺失会损害染色质转录的靶向，从而降低Pol II在转录伸长中的参与度，从而导致Pol II的启动子近端积累。在哺乳动物中，CE在NELF的帮助下抵消转录抑制[28，82]。哺乳动物RNMT-RAM也促进Pol II依赖性转录。RNMT-RAM对转录的影响是直接的并且独立于mRNA加帽、稳定性和翻译。RNMT-RAM结合早期mRNA的整个长度并招募与转录相关的蛋白质。因此，已经发现加帽酶调控转录，并且该调控独立于其酶活性。这种效应的机制似乎是基因和物种特异性的。加帽-转录相互作用的另一个重要参与者是CBC。它与早期转录活动也具有互惠关系。CBC参与转录因子的招募和转录的调节过程。CBC与酵母中的CTD激酶相互作用，其消耗影响CTD Ser2和Ser5的磷酸化水平。在哺乳动物中，CBC通过NELF和P-TEFb的募集刺激转录延伸，并且对CTD Ser2磷酸化很重要。在酿酒酵母中，CBC招募Ctk2或Bur2（P-TEFb的直系对照）。Abd1和CBC对于激酶Ctk1和Bur1的募集很重要，这两者促进了延伸和CE释放。mRNA CBC还通过其与酿酒酵母体内Mot1p的相互作用刺激启动子处的预启动复合物的形成。Mot1p是一种转录调控子，可积极或消极地以一种基因特异性方式调节Pol II转录基因表达。CBC通过阻碍终止因子Pcf11p和Rna15p（裂解因子CFIA的亚基）的募集来抑制弱终止子。因此，CBC在转录过程中具有多重功能。最近，聚腺苷酸结合蛋白相互作用蛋白2被描述为CBC的一种新型伴侣。这些蛋白在转录的早期延伸共同作用，它们的消耗会影响果蝇中的Ser5 CTD Pol II磷酸化。5.加帽作为基因表达的调控步骤近年来，已经出现了一些例子说明mRNA帽状结构形成受到不同信号的调节。这些信号调节mRNA帽状结构形成的速率和程度，导致基因表达的变化。第一个被描述的调节加帽的例子是c-myc依赖性活化。c-Myc调节许多转录子的帽状结构形成。c-Myc增加了催化活性CE向Pol II及其靶基因的募集。此外，c-myc导致帽状结构甲基化增加，这对c-myc依赖性基因调控做出了重大贡献，并且增加的帽状结构甲基化与TFIIH和P-TEFb活性的c-myc依赖性增强有关。由于7-甲基鸟苷帽状结构是有效翻译所必需的，因此增强的甲基化帽状结构形成也将蛋白质产量从c-myc响应基因水平的提高到超过转录诱导的水平。因此，无论转录增强本身如何，加帽的调节都是增强蛋白质合成的一种方式。另一个例子是E2F1转录因子，它通过依赖于Pol II磷酸化的机制促进帽状结构的形成。加帽由信号通路调节。例如，RNMT在细胞周期中被CDK1-细胞周期蛋白B1磷酸化并激活。这导致帽状结构甲基转移酶活性升高，同时转录在有丝分裂后G1期开始时重新开始。这是协调mRNA加帽与转录爆发的方法。在酵母中，营养饥饿导致帽状结构甲基化普遍减少。据报道，输入蛋白α刺激一般帽状结构甲基化。相反，CE mRNA-cap通过调节果蝇中的PKA活性来正向调节Hh信号活性。因此，mRNA加帽的调控因子，加上加帽质量控制机制，也许提供了另一层基因表达的调控。6.结论和前景上述加帽检查点模型已在许多研究中得到证实。然而，目前的加帽/转录相互作用模型尚未完成。有一些问题需要进一步调查。未来一个重要的挑战是澄清将加帽与转录暂停和延伸联系起来的因果关系。加帽蛋白和转录机器之间的广泛相互作用网络尚不清楚。因此，对新生mRNA进行CBC募集的机制知之甚少，监测去帽机器的募集也是如此。物种和基因特异性相互作用特别令人感兴趣。加帽和转录机器具有一些物种特异性特征，例如存在相当数量的后生动物特异性因子和种内副群（表1）。此外，高等真核生物CE的非催化调控域具有高度的多样性，即使在密切相关的物种中。这些事实可能意味着，在与转录装置的特定相互作用中，这些因子和结构域都有参与。上面一些例子提到了物种特异性连接，包括哺乳动物/酵母CE与磷酸-CTD Pol II相互作用的不同位点，芽殖/裂变酵母中GT-RT与Pol II相互作用的不同方式，哺乳动物中存在NELF-CE相互作用，以及转录机制中Abd1 / Pcm1的不同靶标。加帽机制功能中的基因特异性已经在一些例子中进行了描述，例如前面提到的c-myc和E2F1依赖性加帽调节。其他基因特异性转录因子也可以以类似的方式起作用并调节局部加帽。加帽因子也可能是基因特异性信号通路的靶标，例如RNMT的NTD，其携带多个修饰位点。加帽步骤并不总是进行至完成。一个重要的尚未解决的问题是，这是否仅仅是加帽过程固有的低效率的结果，还是调节早期mRNA加工的调控活动。如果后者是正确的，那么所涉及的成分是什么，以及如何做出决策：哪些早期mRNA应该加帽？先前的加帽模型表明，加帽通过阻止mRNA降解，在调控因表达方面起着“被动”作用。现在很明显，加帽可以被修改为“主动”调节mRNA或选定的子集的命运。目前尚不清楚哪组基因和转录因子参与调节加帽。需要更多的研究来更深入地了解信号通路对加帽的影响以及加帽调控在整个基因表达中的作用。原文来源：Interplay of mRNA capping and transcription machineries.