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Letters to the Editor
Mixed-lineage leukemia protein modulates the
loading of let-7a onto AGO1 by recruiting RAN
The mixed-lineage leukemia (MLL) proto-oncogenic protein, as the founding member of human TrxG pro- teins, was originally identified through its association with both acute lymphoblastic leukemia and acute myeloid leukemia.1 MLL is a histone H3 lysine 4 (H3K4) methyltransferase that can execute methylation on a sub- set of target genes through its evolutionarily conserved SET domain, an activity that is essential for normal MLL function.2 MLL is proteolytically cleaved into two distinct subunits: MLLC180 and MLLN320, which non-covalently interact to assemble an intramolecular complex involved in epigenetic transcriptional regulation.2
MLL is routinely regarded as a nuclear protein. Interestingly, however, our recent research revealed that the MLLC180 subunit alone can localize to cytoplasmic processing bodies (P-bodies),3,4 where microRNA (miRNA)-mediated gene silencing takes place,5 and affect the function of a subset of miRNA, as exemplified by the let-7a family.3,4 The dysregulated function of let-7a result- ing from the reduced expression of MLLC180 was very important for maintaining a high level of MYC in MLL leukemia.4 Thus, our work uncovered an unexpected role for MLL in miRNA-mediated translational repression. However, how MLL participates in the regulation of miRNA function remains elusive. We therefore sought to uncover the underlying mechanisms of how MLL partic- ipates in miRNA-mediated translational repression. In this study, we demonstrated that MLL was required to recruit let-7a and miR-10a to the miRNA-induced silenc- ing complex (miRISC), partly through its binding partner RAN. The methods and datasets are available as Online Supplementary Information files.
Most miRNA are loaded onto Argonaute (AGO) pro- teins in the miRISC and act as post-transcriptional regu- lators of their target mRNA.6 Unfortunately, how these miRNA are selectively loaded onto AGO proteins still remains poorly understood.6 Among miRISC-associated factors, AGO1 plays a predominant and specific role in miRNA-mediated translational repression.7 Our immuno- fluorescence results demonstrated that AGO1 and MLL were localized in the same cytoplasmic foci, which was disrupted upon MLL depletion (Figure 1A and B, Online Supplementary Figure S1A-C), suggesting an interaction between AGO1 and MLL. Using a specific P-body marker DCP1A, we further confirmed that MLL and AGO1 co- localized in the cytoplasmic P-bodies (Online Supplementary Figure S1D). Previous studies showed that Argonaute proteins could accumulate in stress granules in addition to P-bodies when cells were subjected to stress.8 We observed that upon arsenite treatment MLL, together with AGO1, could co-localize to stress granules, as indi- cated by the specific stress granule marker G3BP1 (Online Supplementary Figure S1E). These results are consistent with those of our previous study showing that MLL was present not only in P-bodies but also in stress granules.3 Co-immunoprecipitation experiments showed that MLLC180 but not MLLN320 interacts with AGO1 (Figure 1C). Additionally, we demonstrated that the interaction between MLLC180 and AGO1 preferentially occurs in the cytoplasm, and not in the nucleus (Figure 1D). Interestingly, the interaction between MLL and AGO1 decreased dramatically after RNase A treatment, as revealed by co-immunoprecipitation assays, indicating that this interaction was an RNA-dependent indirect interaction, rather than a direct protein-protein interac-
tion (Figure 1E, Online Supplementary Figure S1F). Indeed, the interaction between MLL and AGO1 was enhanced by co-transfected let-7a (Figure 1F, Online Supplementary Figure S1G), indicating that miRNA might play a critical role in the MLL and AGO1 axis.
miRNA-mediated gene silencing requires miRNA to associate with AGO proteins and other silencing factors to form a functional miRISC to repress target mRNA.6 Given that miRNA may fully function in mediating gene silencing even without the existence of microscopically visible P-bodies,9 functional miRISC may still be formed upon MLL depletion. We thus further examined whether the depletion of MLL would impair the recruitment of miRNA to form the functional miRISC. We focused on let-7a and miR-10a, which were two MLL-binding miRNA reported in our previous studies.3 We performed anti-AGO1 RNA immunoprecipitation (RIP) experiments and the results showed that MLL depletion resulted in the loss of binding of let-7a and miR-10a to AGO1 (Figure 1G, Online Supplementary Figure S1H-K). A pull-down assay using biotinylated let-7a further validated that the binding of AGO1 to let-7a was reduced in Mll knockout (Mll-/-) murine embryo fibroblasts (MEF) (Figure 1H). In addition, the recruitment of let-7a and miR-10a target mRNA, MYC, HRAS and HOXA1, to AGO1 was largely impaired in the MLL-depleted cells (Figure 1I and Online Supplementary Figure S1L and S1M). Notably, AGO1 expression was not affected by the knockdown of MLL (Online Supplementary Figure S1H), suggesting that this impaired recruitment of miRNA and its target mRNA to AGO1 was likely not caused by the reduced AGO1 pro- tein levels. Together with the above-mentioned data showing that the interaction between MLL and AGO1 was RNA-dependent, these results indicated that MLL and miRNA may require each other in order to be effi- ciently recruited by AGO1 and form a functional miRISC.
To further investigate the role of MLL in the recruit- ment of miRNA to miRISC, we reintroduced shRNA- resistant MLLN320, MLLC180 or full-length MLL (MLLFL) into MLL knockdown 293T cells or Mll knockout (Mll-/-) MEF cells and found that the recruitment of let-7a and miR-10a to miRISC was rescued by exogenous MLLC180 (Figure 1J and K, Online Supplementary Figure S1P-S). Collectively, these results indicated that MLL plays a causal role in tar- geting miRNA and their target mRNA to AGO1 to form a translationally repressed miRISC complex, highlighting the importance of MLL in the control of miRNA-mediat- ed expression.
MLLC180 itself does not possess any predictable RNA recognition motif, so we reasoned that MLL might recruit RNA components indirectly through its binding partners. Our proteomics data showed that RAN, a small GTPase involved in the import of cargo through nuclear pore complexes,10 was one of the proteins displaying strong interactions with MLL in the cytoplasm (Figure 2A). In line with a previous study,11 we found that RAN interact- ed with MLL in an RNA-independent manner (Figure 2B). We further confirmed that RAN could pull down MLLC180, indicating a direct interaction between MLLC180 and RAN (Figure 2C). Moreover, immunofluorescence data showed that upon arsenite treatment, MLL together with RAN co-localized to stress granules, as revealed by a stress granule marker eIF3 (Figure 2D), suggesting a potential role of RAN in regulating mRNA translation besides involving the import of cargo. RAN and XPO5 can form a complex which plays a critical role in nucleocytoplas- mic transport of pre-miRNA molecules.10 Unlike XPO5, which dissociates from pre-miRNA in the cytoplasm, RAN could still associate with pre-miRNA in the cyto-
haematologica | 2021; 106(7)
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