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.
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