MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Abstract

Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking ("CLIP" and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP ("TRIBE"). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β -actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the β -actin gene. This result indicates that MCP-ADAR bound to β -actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.3 Introduction:

TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

“…In summary, TRIBE works well for eIF4E-BP, in mammalian cells and in Drosophila cells. Recent results from elsewhere on different RBPs show that TRIBE also works well in other mammalian systems (40)(41)(42). Moreover, our current results suggest that TRIBE demonstrates more target transcript specificity than CLIP.…”

“…Moreover, our current results suggest that TRIBE demonstrates more target transcript specificity than CLIP. Although work in this paper does not constitute a proper head-to-head comparison of TRIBE and CLIP, this suggestion is similar to conclusions from other TRIBE versus CLIP comparisons in mammalian cells (41). Because ADAR deamination is slow (17), TRIBE editing may reflect targets with longer RBP dwell time and therefore tighter binding, whereas cross-linking may be better able to capture ephemeral associations between a RBP and RNA.…”

TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression

“…TRIBE target genes that are also identified with other methods are less likely to be false positives and may reflect RNAs bound at high affinity by the RBP (22,34). By comparison, TRIBE is an antibody-independent method, and RIP and CLIP targets may include false positives due to intrinsic antibody issues (49). The shorter TRIBE gene list may also reflect more false negatives, due perhaps to inefficiencies of TRIBE as previously discussed (22).…”

“…Activation of CREB alleviated the defects in dendritic morphology caused by TDP-43 dysfunction, thus validating our use of TRIBE to identify TDP-43 targets relevant to cellular function. In addition, TRIBE was recently performed for other mammalian RBPs (49,50), indicating that TRIBE will have wide applicability.…”

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

“…Another RNA-based system where a reliable design algorithm can help bring about the full potential of the technology is the encoding of multiple repeats of phage coat protein binding elements on an RNA molecule of choice. In particular, the latter technology has been utilized in many studies and proven to be an important tool in gene editing and RNA-tracking related applications [7][8][9][10][11][12] . However, current cassettes are characterized by repeated sequence elements, making them nearly impossible to synthesize, clone, and maintain, which limits their utility for robust quantitative measurements 13 as well as expansion to more complex multi-genic applications.…”