Regulation of pluripotency by RNA binding proteins.
|核心内容：
建立、维持和退出多能性需要细胞分子机制的精确协调。
在破译这一复杂系统的许多方面，特别是在表观遗传学、转录和非编码RNA方面，已经取得了实质性的进展。
对转录后调控过程的关注较少，如选择性剪接、RNA加工和修饰、核输出、转录本稳定性的调节和翻译。
在这里，我们介绍RNA结合蛋白，这些蛋白能够实现基因表达的转录后调控，总结当前和正在进行的关于它们在不同调控点的作用的研究，并讨论它们如何帮助决定多能干细胞的命运。原文摘要：
Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery.
Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs.
Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation.
Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells.
Introduction Embryonic stem cells (ESCs), which are derived from the inner cell mass of the mammalian blastocyst, are remarkable because they can propagate in vitro indefinitely while retaining both the molecular identity and the pluripotent properties of the peri-implantation epiblast.
Consequently, ESCs provide a biologically relevant and experimentally tractable model system for studying regulators of cell fate and cell fate transitions in early development.
Understanding the molecular mechanisms of ESC maintenance and differentiation is critically important not just scientifically but also clinically, because an improved knowledge of pluripotency and embryonic development will allow ESCs to be more effectively utilized as an in vitro platform for disease modeling, drug discovery, and tissue regeneration.
While the transcriptional, signaling, and epigenetic regulation of these cells have been the primary focus of research efforts in recent years , posttranscriptional and translational mechanisms of control remain relatively unexplored, despite evidence that they play a dominant role in driving ESC fate decisions.
Indeed, posttranscriptional regulation has been reported to account for nearly 75% of the changes in protein levels after differentiation induced by knockdown of the transcription factor Nanog (Lu et al., 2009), and it was recently demonstrated that control over translational initiation by the eIF4e binding proteins dramatically influences the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs) (Tahmasebi et al., 2014).
The cell controls protein levels posttranscriptionally using a large collection of tools that includes noncoding RNAs and RNA binding proteins (RBPs).
Recent work elucidating the functions of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in ESCs has been comprehensively reviewed elsewhere.
Similarly, posttranslational regulation of protein levels through the addition of covalent modifications also has been discussed recently (Wang et al., 2014b).
The purpose of this Review is specifically to address the roles of RBPs in the maintenance and differentiation of ESCs.
RBPs are responsible for every event in the life of an RNA molecule, including its capping, splicing, cleavage, nontemplated nucleotide addition, nucleotide editing, nuclear export, cellular localization, stability, and translation (Keene, 2007).
Overall, little is known about RBPs: most are classified based on computationally predicted similarities to proteins with known RNA binding domains, and until recently, few of these predictions have been verified in a cellular context in vivo.
The recent introduction of a technique termed ''mRNA interactome capture,’’ which enables the identification of proteins bound to polyadenylated RNAs in vivo, has been a significant development for the field (Baltz et al., 2012; Castello et al., 2012).
Using this method, several groups were able to create a comprehensive catalog of RBPs in different mammalian cells, including 555 RBPs in mouse ESCs (Kwon et al., 2013).
However, the functions of these RBPs in ESCs and their changes in ESC differentiation have yet to be addressed.
Indeed, the mechanism of action of only a small number of RBPs has been examined in any great detail in the context of pluripotency.
Here, we summarize current knowledge of RBP contribution to posttranscriptional and translational regulation in ESCs, following the approximate order that each regulatory event would be encountered as a transcript born in the nucleus migrates into the cytoplasm and is translated into a polypeptide (diagrammed in Figure 1).
Figure 1. RBPs Involved in Pluripotency Act at Many Different Regulatory Steps
Summary of the RBPs and the events they regulate in the maintenance and exit from pluripotency as discussed in this Review.
Starting in the nucleus, RBPs regulate splicing (FOX2, SON, SFRS2, MBNL1, and MBNL2) and alternative polyadenylation (FIP1) simultaneously with transcription.
RBPs then regulate export of transcripts (THOC2 and THOC5).
RBPs also can induce modifications to RNAs including nucleotide changes (ADAR, METTL3, and METTL14 in nucleus) and nucleotidyl transfer (LIN28A in association with the TUTases ZCCHC6 and ZCCHC11 in the cytoplasm), which in turn influence mRNA stability and translation.
In the cytoplasm, the binding of RBPs to the 3' UTRs of transcripts directly regulates mRNA stability and translation (TRIM71, PUM1, and BRF1).
Translation is also influenced by RBPs that bind the 5’UTR of transcripts (NAT1, RBM35A, and PTBP1).
Blue circles indicate RBPs. RBP genes in red are positive regulators of pluripotency.
RBP genes in green are negative regulators of pluripotency.
Black circles indicate the protein products of the genes whose expression levels are affected by RBPs.
Throughout, we also discuss potential directions of future inquiry that will allow us to more fully appreciate the scope of RBP-mediated posttranscriptional and translational regulation in pluripotency.
● Regulation of Translation ●
RBPs can also directly modulate protein translation, often through binding at the 5’UTR of RNA transcripts.
In so doing, RBPs recruit translation initiation factors, adjust the accessibility of the RNA to ribosomes, create ribonucleoprotein structures conducive for cap-independent and internal ribosome entry site (IRES)-mediated translation, and regulate the movement of the ribosomes along the transcript.
conducive: If one thing is conducive to another thing, it makes the other thing likely to happen.
Given these many roles, it is no surprise that RBPs involved in these 5'UTR-related processes have also been linked to the control of ESC pluripotency.
In particular, RBP-5' UTR interactions involving the proteins Nat1, Rbm35a (Esrp1), and Ptbp1 have been shown to regulate ESC differentiation and proliferation.
Nat1 was first identified in liver carcinomas as a general repressor of translation.
In mouse ESCs, depletion of Nat1 does not affect proliferation and self-renewal in ESC growth conditions, but Nat1-/- ESCs exhibit a defect in retinoic acid (RA)- induced differentiation (Yamanaka et al., 2000).
Previous studies in other cell lines have shown that Nat1 is homologous to the translation initiation factor eIF4G, is located in the cytoplasm and can autoregulate its own translation from an IRES in its mRNA; thus, the authors speculated that Nat1 binds to highly structured 5'UTRs and associates with translational initiation factors.
However, their study did not detect a difference in cap-dependent or cap-independent translation in wild-type and Nat1-/- ESCs as measured by [35S]methionine incorporation and bicistronic luciferase assays, respectively.
然而，他们的研究分别通过[35S]蛋氨酸掺入法和双反子荧光素酶试验并没有检测到野生型和Nat1-/- ESCs中cap依赖或cap不依赖翻译的差异。
Despite the negative results, it is important to note that these experiments were conducted under steady-state pluripotency conditions, not under the differentiation conditions in which they observed the phenotype.
Therefore, it remains plausible that Nat1 influences ESC differentiation through translational mechanisms, and further studies should address the role of Nat1 in transitions into and out of pluripotency.
plausible[ˈplɔːzəbl] An explanation or statement that is plausible seems likely to be true or valid.
Like Nat1, Rbm35a is a negative regulator of pluripotency.
Knockdown of Rbm35a inhibits ESC differentiation and promotes somatic cell reprogramming by increasing expression of Oct4, Nanog, and Sox2.
Indeed, Rbm35a immunoprecipitation and polysome profiling show that Rbm35a normally binds to the 5'UTR of Oct4 and Sox2 mRNAs, thus preventing them from being loaded into polysomes (Fagoonee et al., 2013).
In addition, Rbm35a activity is not restricted to ESCs, because the RBP is a regulator of alternative splicing in epithelial cell lines (Warzecha et al., 2009) and has also been shown be expressed in tumor cells where it binds to highly structured GC-rich 5' UTRs of oncogenes and prevents their translation (Leontieva and Ionov, 2009).
ESCs themselves form teratomas when injected into mice and share many properties with somatic tumors, including limitless replicative potential.
teratomas/ˌtɛrəˈtəʊmə/ a tumour or group of tumours composed of tissue foreign to the site of growth
ESCs本身在注射到小鼠体内后形成畸胎瘤，并与体细胞肿瘤有许多相似的特性，包括无限的复制潜能。
Therefore, Rbm35a suppression may be an important component of maintaining ESC and tumor cell immortality[ˌɪmɔːrˈtæləti] （immortal，long live）through both shared and unique molecular targets present in these different cellular contexts.
Ptbp1, another RBP that binds the 5'UTR of gene transcripts, also appears to control ESC growth through its regulation of the cell cycle.
Ptbp1 knockout ESCs have a proliferation defect with a prolonged G2/M phase.
This phenotype appears to be at least in part secondary to problems with chromosomal segregation.
Bicistronic luciferase assays show that Ptbp1 binds to the IRES of CDK11p58 and represses translation of this gene, high levels of which are associated with a prolonged telophase caused by chromosomal lagging.
双顺反子荧光素酶分析表明，Ptbp1与 CDK11p58的 IRES 结合，并抑制该基因的翻译，其(CDK11p58)高水平与染色体滞后引起的延长的末期相关。
Overexpression of CDK11p58 in wild-type ESCs led to chromosome missegregation (Ohno et al., 2011).
Nevertheless, it is unclear whether or not overexpression of CDK11p58 is the sole contributor to the Ptbp1 knockout phenotype—a question that could be addressed through global comparisons of mRNA and protein abundance in wild-type and knockout cells.
Notably, Ptbp1 has also been extensively studied in other cell lines, where it has been implicated in alternative splicing, alternative polyadenylation, mRNA stability at the 3'UTR, and IRES-driven translation (Boutz et al., 2007; Castelo-Branco et al., 2004; Kosinski et al., 2003; Bushell et al., 2006).
It would be useful and informative for future studies to examine whether these other well-known functions of Ptbp1 are also involved in pluripotency.
● Conclusions and Perspectives ●
It is without a doubt that posttranscriptional regulation by RBPs contributes extensively to the establishment and maintenance of, as well as exit from, the ESC state.
The field remains relatively young and largely uncharted, however, and there are many opportunities for further inquiry and discovery.
chart A chart is a diagram, picture, or graph which is intended to make information easier to understand.
As described above and summarized in Table 1, a number of studies have documented the significance of individual RBPs in pluripotency based on knockdown and knockout models demonstrating that perturbation of the RBP disrupts the wildtype ESC phenotype.
However, few probe the actual mechanism by which the RBPs produce their effect in ESCs.
Instead, RBP function has been examined mostly in cancer cell lines and cell-free biochemical assays, the in vivo relevance of which needs to be more clearly characterized.
The studies that do delve into the molecular mechanisms in ESCs generally examine only a limited number of targets.
delve[delv] into: pick , probe into,tunnel , probe into;
While the RBPs discussed here are not unique to ESCs, cell context likely influences their downstream effects on cell fate and cell fate transitions significantly by providing a specific combination of intracellular pathways with which these RBPs interface.
interface If one thing interfaces with another, or if two things interface, they have connections with each other. If you interface one thing with another, you connect the two things.
Thus, to reach a deep and unified understanding of the role of RBPs in pluripotency, we will need to make use of global approaches like those that have already been successfully applied to the field of epigenetics.
In particular, we must dissect all RBP-RNA interactions systematically in ESC self-renewal and differentiation conditions so as to examine not only how RNA transcript levels change but also how the components of ribonucleoprotein (RNP) complexes are rearranged during transitions from one cell fate to another.
核蛋白（英文：nuclear protein）是指在细胞质内合成， 然后运输到核内起作用的一类蛋白质。如各种组蛋白、DNA合成酶类、RNA转录和加工的酶类、各种起调控作用的蛋白因子等。核蛋白一般都含有特殊的氨基酸信号序列， 起蛋白质定向（远程联系还是布朗运动碰撞？）、定位(抓手)作用。
Intriguingly, as mentioned above under the subsection ''Multifunctional RBPs,’’ a number of RBPs appear to be involved in multiple aspects of RNA metabolism in both the nucleus and the cytoplasm.
This observation may result from an RBP having different roles in different cell types, multiple roles in a single cell type, or some mix of the two.
Regardless, it is almost certain that the particular combination of targets and cofactors that an RBP encounters influences its functions in context-specific ways—a notion that expands upon the ''RNA regulon’’ model originally proposed by Jack Keene, in which an RBP binds multiple targets to effect changes in various cellular processes (Keene, 2007).
无论如何，几乎可以肯定的是: RBP 遇到的特定目标和辅助因子的组合会以特定上下文的方式影响它的功能ーー这个概念扩展了 Jack Keene 最初提出的“ RNA 调控”模型，在这个模型中 RBP 与多个目标结合，从而影响各种细胞过程的变化。（简单理解就是RBP与不同的辅助因子结合形成不同的复合物，不同复合物可以结合不同的靶标，并行使不同的功能；RBP 形成的同一复合物在同一细胞的不同生理状态或不同的细胞里可能有不同的功能。 ）
In other words, it is possible that instead of regulating multiple targets through just a single mechanism, one RBP could simultaneously participate in several layers of RNA metabolism (Figure 2).
Figure 2. The Multifunctional RBP: The RNA Regulon Revisited
(A and B) Alternative models for RBP regulation of RNA metabolism.
(A) In the classical view of the RNA regulon, an RBP (blue object) binds multiple transcripts to execute a single action on many RNAs (purple, blue, and green). This in turn can affect an array of cellular processes depending on the nature of the mRNAs targeted.
(B) In an expanded version of the RNA regulon model, an RBP not only has multiple targets but also acts on those targets at multiple levels of intracellular RNA metabolism. An example RBP shown here regulates transcription, splicing, RNA stability, and mRNA translation of a common set of transcripts. While it is unlikely that such an RBP exists, we propose that many RBPs will have a subset of these functions on overlapping sets of targets.
In so doing, the RBP becomes part of increasingly complex regulatory modules, with many opportunities for feedback and crosstalk, where one aspect of metabolism such as translation can be linked to another such as splicing.
feedback：
could be positive or negative，positive feedback to adapt/response to environment alteration、cell fate change、reprogramming、programmed cell death；negative feedback to retain cell homeostasis；
crosstalk：imagine signal cascade as line or flux, crosstalk will reinforce the signal network；
Consequently, perturbation of the RBP could have a cascading effect on the molecular landscape of a cell and precipitate drastic switches in cellular identity.
drastic[ˈdræstɪk: fierce , acute , violent , hard , furious,extreme
Also, it follows that disrupting subsets of the mRNA targets or cofactors of any one RBP could affect parallel pathways by shifting the RBP’s dominant activities to different genes or even different regulatory modules altogether.
For example, a decrease in the levels of a cofactor that enables an RBP to regulate splicing could drive that RBP to shift its main activity to transcription.
Moving forward, it will be critical to investigate RBPs at the genomics level in biologically relevant cell types, focusing on those states in which the RBPs are demonstrated to play significant roles.
Studying RBPs in ESCs provides an excellent starting context to achieve such a goal, because not only can ESCs provide unlimited untransformed material for large-scale genomic studies, they can also be differentiated down any cellular lineage to determine how context changes protein function.
Thus, experimental tools and platforms developed in the ESC system can be used to study a multitude of cell types that comprise the mammalian body plan.
-tude :SUFFIX forming N indicating state or condition 表示“状态”，“条件”
参考文献：http://stem-art.com/Library/Science/Regulation%20of%20Pluripotency%20by%20RNA%20Binding%20Proteins.pdf
