TRIM71 Binds to CRD Complex Members and Is Capable of Positive and Negative Regulation of Target RNAs

Abstract

TRIM71 is an important RNA binding protein in development and disease. It is expressed early in development and plays a role in promoting early cell fates and preventing premature differentiation of cells. TRIM71 was discovered in C. elegans as a target of the let-7 microRNA and it is conserved in humans. Its expression during development is largely controlled post-transcriptionally by let-7 and it is downstream of the LIN28/let-7 axis. TRIM71 has been described as inhibiting several target mRNAs such as CDKN1A and EGR1. To better understand the role of TRIM71 in cells and the RNAs it binds, we used crosslinking IP (CLIP) followed by RNA sequencing. We discovered a number of developmentally and disease relevant RNAs that were bound by TRIM71 such as LIN28B, MYC, MDM2, and the MBNL and NFI families. To understand how TRIM71 regulated RNAs in a cell we analyzed proteins that co-precipitated with TRIM71. TRIM71 was able to co-precipitate members of the CRD complex which is known to bind to and positively regulate RNAs such as MYC. Domain deletion analysis of TRIM71 showed that the coiled-coil domain was necessary for the interaction with DHX9, but we could not narrow down the domains interacting with the core CRD subunit, IMP1. However, the RNA-binding NHL domain of TRIM71 did not alone bind the CRD complex members. To determine the intrinsic RNA binding abilities of TRIM71 we depleted IMP1 and showed that TRIM71 was able to bind RNA more promiscuously. Additionally, we performed a CLIP-Seq of the NHL domain alone and similarly found more promiscuous binding compared to wild-type TRIM71. We concluded that the CRD complex binds to TRIM71 and promotes specificity of RNA binding. This new work shows that TRIM71 can positively regulate expression of genes with key roles in oncogenesis. We therefore analyzed the importance of TRIM71 in cancer, finding that its expression correlates with LIN28A/B in several cancer types. One of these, hepatocellular carcinoma, showed decreased cell proliferation and increased senescence when TRIM71 was depleted. This work shows that TRIM71 and LIN28A/B, in addition to being linked in development, can also be linked in oncogenesis.