Proc Natl Acad Sci U S A . 2020 Sep8;117(36):22068-22079.
doi: 10.1073/pnas.2006617117. Epub 2020 Aug 24
Background
RNA–protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA–protein interactions in living cells in an unbiased manner. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. The interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA–protein interactions in living cells. Mapping networks of RNA–protein interactions in living cells is necessary to enable a mechanistic understanding of RNA processing, trafficking, folding, function, and degradation. Many protein-centered approaches and methods represent an important technological advance and have yielded biological insights. However, the long labeling time window of BioID is not optimal for the study of dynamic processes (for example, rapid changes in RNA interactomes in response to cellular stress), while the stem-loop tags could affect the function of target RNAs.
Methods
Methods relate to cloning, Western blots, proteomic sample preparation and analysis, additional data analysis, confocal fluorescence imaging, m6A pulldown assay, and telomerase activity assay.
Results
Using MS2-MCP to Target APEX to Tagged hTR. We utilized two complementary approaches to deliver APEX to the site of hTR for proximity labeling. The first entails conjugating hTR to the bacteriophage MS2 RNA stem loop, which can specifically bind an MS2 coat protein-fused APEX2 (MCP- APEX2) with high affinity (Kd < 1nM) (34). In the second approach, a catalytically inactive Cas13-APEX2 fusion (dCas13-APEX2) is programmed using a guide RNA (gRNA) to target unmodified hTR, but with a lower binding affinity (Kd ~ 10nM).Development of a CRISPR-Cas13 Strategy to Target APEX to Untagged hTR.Proteomic Mapping of hTR Interacting Partners via APEX Proximity Labeling. To further analyze the 49 proteins we enriched in three or more datasets, we performed clustering based on prior protein–protein interaction evidence in the STRING database (67) (Fig. 3E). The largest cluster contains almost exclusively RNA binding proteins (15 out of 18), including the key telomerase component DKC1, the hTR degradation complex component DGCR8, and the core Cajal body component COIL. Other smaller groups that closely associate with the major cluster also contain numerous proteins related to hTR, such as the Cajal body component ICE2.ALKBH5 Binding to hTR Regulates Telomerase Activity through m6A Modification.
To validate our proteomic identification of ALKBH5 as a possible interaction partner of hTR, we performed an immunoprecipitation experiment. We note that published ALKBH5CLIP-seq data (76) exist only for polyadenylated RNAs, not noncoding RNAs such as hTR. RT-qPCR analysis in Fig. 4A shows that immunoprecipitation of FLAG-tagged ALKBH5 enriches endogenous hTR but not a different nuclear noncoding RNA, HOTAIR. We next asked whether endogenous hTR is directly modified by m6A. A RIP experiment using anti-m6A antibody gave enrichment of hTR compared to negative controls (Fig. 4B). We further tested if overexpression of ALBKH5 reduces m6A levels on hTR; indeed, anti-m6A RIP enriches less hTR RNA when ALKBH5 is overexpressed, suggesting that ALKBH5 activity can reduce the levels of m6A on hTR (Fig. 4C). A control experiment showed that the total level of hTR was unchanged upon ALKBH5 overexpression (Fig. 4D).
Conclusions
We developed two complementary methods for tagging endogenous proteins in the vicinity of specific cellular RNAs, for subsequent identification by mass spectrometry. When applied to the human telomerase RNA, our methods recovered known interaction partners as well as unexpected hits, including an enzyme that catalyzes RNA posttranscriptional modification to influence telomerase activity. The technology introduced by our study should facilitate future investigations into RNA–protein interactions in living cells.