Integration of the results from systems-level determination of cis-acting elements into high-resolution maps of posttranscriptional regulatory events.

我们最近发现了一对深入研究的、不相关的RNA结合蛋白家族，Igf2BP和Lin28，它们在靶mRNAs上有重叠的结合部位，因此构成了研究转录后基因调控中协同或竞争的有吸引力的候选。哺乳动物Lin28a和LIN28B是RBP家族的成员，在动物体内保守，含有两个CCHC ZF结构域和一个冷休克结构域(CSD)。最初，LIN28基因被鉴定为线虫发育中的异时性基因，在人类中，这些基因也在胚胎组织中阶段特异性表达，在较小程度上，在成人组织中表达。LIN28蛋白调控哺乳动物发育的各个方面，主要归因于对在动物发育中起关键作用的let-7miRNA家族大多数成员的直接抑制。最近，我们的合作者Stefan Muljo博士(整合免疫学单位，NIAID)描述了通过过表达Lin28b(袁，2012)，成年小鼠的造血干细胞(HSC)重新编程为类似胎儿的状态。有趣的是，胎儿HSC表现出高水平的LIN28表达，同时仍然保持着唯一的LIN28独立的let-7家族成员的一些表达，这表明LIN28在基因调控中发挥了额外的作用。我们和其他人之前一直发现LIN28蛋白与数千个mRNA相互作用。然而，异位表达的LIN28对其mRNA靶标的调控作用和分子机制仍然不清楚，可能是因为在所研究的细胞系中缺乏特定的辅助因子。我们想用造血细胞作为一个模型系统来剖析这个多功能RBP家族的let-7独立的分子机制，并将Lin28a和B靶标定位在B细胞前体细胞系(220-8)中。我们发现数千个结合位点分布在5,000个mRNAs的CDS和3UTR上，不出所料，LIN28在该细胞系中的表达也导致了miRNA let-7家族的抑制，同时也导致了220-8中表达的328个保守的let-7靶向mRNAs的强烈反转录。然而，LIN28的直接mRNA靶点累积到更高的水平，前1,403个LIN28靶点的mRNA水平累积超过2倍，表明LIN28的大部分作用是直接的，不依赖于LET-7的抑制。我们的观察结果与之前的研究发现对LIN28靶点几乎没有直接调控作用形成鲜明对比，表明前B细胞系代表了一种适合于研究miRNA非依赖性调控LIN28靶点的系统。使用无偏倚的蛋白质组学方法，我们确定了RBP IGF2BP3与LIN28的直接相互作用。这种相互作用不依赖于RNA，对220-8个IGF2BP3结合位点的定位表明LIN28和IGF2BP3结合位点有显著重叠。有趣的是，IGF2BP家族蛋白与LIN28s癌胎儿表达模式相同，我们之前已经证明这会导致他们的目标mRNAs适度稳定。最重要的是，我们发现了通过IGF2BP3和LIN28与重叠位点结合来协同稳定靶mRNAs，据我们所知，这构成了来自不同家族的两个限制性商业惯例合作放大各自对mRNA靶的影响的第一个例子。在未来，我们将与Muljos博士一起进行功能获得性基因回溯实验，以确定IGF2BP1-3是否可能与Lin28a/B合作确定胎儿HSC的命运。如果初步结果是有希望的，我们将使用CRISPR/Cas9基因组编辑技术来产生三重突变的IGF2BP1-3小鼠。为了实现这些目标，我们将使用Hafner实验室所有项目的结合谱数据来识别具有重叠或互斥结合谱的RNA结合蛋白对，以便进一步研究RNA结合蛋白的竞争和协同作用。

We recently identified a pair of intensely studied, unrelated RNA binding protein families, Igf2bp and Lin28, with overlapping binding sites on target mRNAs and thus constituting attractive candidates for investigation of synergy or competition in posttranscriptional gene regulation. Mammalian LIN28A and B are members of an RBP family conserved in animals containing two CCHC ZnF domains and one cold-shock domain (CSD). Initially LIN28 was identified as a heterochronic gene in C. elegans development, and in humans these genes are also stage-specifically expressed in embryonic tissues and, to a lesser extent, in adult tissues. That LIN28 proteins regulate various aspects of mammalian development was mainly attributed to a direct repression of most members of the let-7 miRNA family with key functions in animal development. Recently, our collaborator Dr. Stefan Muljo (Integrative Immunology Unit, NIAID) described reprogramming of adult hematopoietic stem cells (HSC) from mice into a fetal-like state by overexpression of LIN28B (Yuan, 2012). Interestingly, fetal HSCs exhibit high levels of LIN28 expression, while still maintaining some expression of the only LIN28-independent let-7 family member, suggesting additional roles for LIN28 in gene regulation. Consistently, we and others previously found that LIN28 proteins interacted with thousands of mRNAs. However, the regulatory effect and molecular mechanism of ectopically expressed LIN28 on its mRNA targets remained elusive, possibly due to the lack of specific co-factors in the cell lines studied. We wanted to use hematopoietic cells as a model system to dissect the let-7 independent molecular mechanisms of this multifunctional RBP family and mapped LIN28A and B targets in a B-cell progenitor line (220-8). We found thousands of binding sites distributing over the CDS and 3UTR of 5,000 mRNAs and, as expected, LIN28 expression in this cell line also resulted in a repression of the miRNA let-7 family with a concomitant robust derepression of the 328 conserved let-7 target mRNAs expressed in 220-8. However, direct mRNA targets of LIN28 accumulated to an even higher level, with the top 1,403 LIN28 targets showing a greater than 2-fold accumulation of mRNA levels, indicating that the bulk of the LIN28 effect was direct and independent of let-7 inhibition. Our observations were in sharp contrast to previous studies finding few direct regulatory effects on LIN28 targets, indicating that the pro-B-cell line represents a suitable system for the study of miRNA-independent regulation of LIN28 targets. Using unbiased proteomic approaches we identified a direct interaction of the RBP IGF2BP3 with LIN28. This interaction was RNA independent and mapping of the IGF2BP3 binding sites in 220-8 revealed a significant overlap of LIN28 and IGF2BP3 bound sites. Intriguingly, the IGF2BP family of proteins shares LIN28s oncofetal expression pattern, which we have previously shown leads to modest stabilization of their target mRNAs. Most significantly, we found a synergistic stabilization of target mRNAs by binding of IGF2BP3 and LIN28 to overlapping sites, which constitutes to our knowledge the first example of two RBPs from different families collaborating to amplify their respective effects on mRNA targets. In the future, together with Dr. Muljos group, we will perform gain-of-function retrogenic experiments to determine whether IGF2BP1-3 might collaborate with LIN28A/B in specifying the fetal HSC fate. If the preliminary results are promising, we will use CRISPR/Cas9 genome editing technology to generate triple mutant IGF2BP1-3 mice. Toward these goals, we will use the binding profile data from all projects in the Hafner lab to identify pairs of RNA binding proteins with overlapping or mutually exclusive binding profiles for further study of competition and synergy of RNA binding proteins.