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被深入研究的、不相关的RNA结合蛋白家族，Igf2bp和Lin28，它们在靶mrna上具有重叠的结合位点，因此是研究转录后基因调控协同或竞争的有吸引力的候选者。哺乳动物LIN28A和B是RBP家族的成员，在含有两个CCHC ZnF结构域和一个冷休克结构域（CSD）的动物中保守。最初，LIN28被确定为秀丽隐杆线虫发育中的异慢性基因，在人类中，这些基因也在胚胎组织中特异性表达，并且在较小程度上在成人组织中表达。研究表明，LIN28蛋白主要通过直接抑制在动物发育中具有关键功能的let-7 miRNA家族的大多数成员来调节哺乳动物发育的各个方面。最近，我们的合作者Stefan Muljo博士（NIAID综合免疫学单位）描述了通过过度表达LIN28B将小鼠成年造血干细胞（HSC）重编程为胎儿样状态（Yuan, 2012）。有趣的是，胎儿造血干细胞表现出高水平的LIN28表达，同时仍然保持唯一的LIN28独立的let-7家族成员的一些表达，这表明LIN28在基因调控中具有其他作用。一致地，我们和其他人先前发现LIN28蛋白与数千种mrna相互作用。然而，异位表达的LIN28对其mRNA靶点的调控作用和分子机制尚不清楚，可能是由于在所研究的细胞系中缺乏特异性的辅助因子。我们想用造血细胞作为模型系统来解剖这个多功能RBP家族的let-7独立分子机制，并在B细胞祖系中定位LIN28A和B靶点（220-8）。我们发现数千个结合位点分布在5000个mrna的CDS和3UTR上，正如预期的那样，该细胞系中LIN28的表达也导致miRNA let-7家族的抑制，同时伴随有328个保守的let-7靶mrna在220-8中表达的强烈抑制。然而，LIN28的直接mRNA靶点积累到更高的水平，前1403个LIN28靶点的mRNA水平积累超过2倍，这表明LIN28的大部分作用是直接的，独立于let-7的抑制。我们的观察结果与以往研究发现的对LIN28靶点的直接调控作用形成鲜明对比，表明pro- b细胞系是研究LIN28靶点的mirna独立调控的合适系统。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。这种相互作用是RNA独立的，对220-8个细胞中IGF2BP3结合位点的定位显示，LIN28和IGF2BP3结合位点有明显的重叠。有趣的是，IGF2BP家族的蛋白共享lin28的癌胎表达模式，我们之前已经表明，这导致了它们的靶mrna的适度稳定。最重要的是，我们发现IGF2BP3和LIN28结合在重叠位点上对靶mRNA具有协同稳定作用，这是据我们所知，来自不同家族的两个rbp合作放大各自对mRNA靶点影响的第一个例子。在未来，我们将与Muljos博士的团队一起进行功能获得性逆转录实验，以确定IGF2BP1-3是否可能与LIN28A/B共同指定胎儿HSC的命运。如果初步结果有希望，我们将利用CRISPR/Cas9基因组编辑技术，产生三突变体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 are 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. LIN28 proteins have been shown to regulate various aspects of mammalian development, mainly via 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 distributed 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 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 that found only a 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 cells 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 the first example of two RBPs from different families collaborating to amplify their respective effects on mRNA targets to our knowledge. 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.