Nucleic Acids Res
January 1, 2021;
FAX-RIC enables robust profiling of dynamic RNP complex formation in multicellular organisms in vivo.
RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1
, NCBP2, EIF4E
, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte
transition, for instance, from canonical eIF4E
to noncanonical eIF4E3
. Furthermore, using Mus musculus liver
, we demonstrate that FAX-RIC is applicable to mammalian tissue
samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.
Nucleic Acids Res
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Graphical Abstract. FAX-RIC profiled distinct RBPs with significantly enhanced efficiency in HeLa cell. FAX-RIC report highly comprehensive RNA interactome profiles in X. laevis oocytes and embryos, and mouse liver tissue.
Figure 1. Development of FAX-RIC and its specificity for the representative RBPs in HeLa cell. (A) Schematic outline of the FAX based RNA interactome capture (FAX-RIC) method. Biological samples are treated with formaldehyde solution in PBS at conditions that are separately optimized to form covalent bond between proximal RNA–protein interactions. RNA crosslinked proteins are enriched through oligo-dT pulldown of poly(A) tailed RNA. Profile of enriched protein samples are obtained via LC-MS/MS analysis. (B) SDS-PAGE and silver staining of the oligo-dT pulldown samples from the lysate of HeLa cells that are treated with indicated formaldehyde and UV light crosslinking conditions. (C) Western blot analysis for representative RBPs (EIF4E, AGO1 and PABP) and a negative control protein (Tubulin A).
Figure 2. FAX-RIC profile known human RBPs with high specificity. (A) Defining the high confidence FAX RNA interactome in HeLa cell. Volcano plot displaying the fold-change of average LFQ intensity (FAX-RIC/NoX-RIC) (x-axis) and the –log10 Student's t-test P value (y-axis) for all the proteins quantified in at least two out of three replicate FAX-RIC experiment. Proteins with log2 fold-change >1 and statistically significant enrichment over the NoX-RIC (P value <0.01, Student's t–test, adjusted by Benjamini–Hochberg method) are highlighted in red. (B) UpSet plot for the number of proteins that are identified in indicated group of RBP profiles obtained from our FAX-RIC in HeLa cell and previous UVX based RBP profiling studies (3–7). Each bar on the plot represent the number of proteins that were identified in single or multiple RNA interactome profiles denoted by the black dots below for respective studies whose name and the number of identified proteins are indicated on the left column. For example, the first and second bar on the plot represent the number of RBPs that were exclusive to Trendel et al. (5) and Queiroz et al. (6) study, respectively. The third bar represent the number of RBPs that were common to all six RNA interactome profile, indicated by the 6 black dots joined by a solid line. Number of the proteins that are exclusive to FAX-RIC and three representative RIC experiments (3,4,7) are highlighted in red or orange, respectively. (C) Number and proportion of proteins annotated with the known RBDs, either ‘classical’ or ‘non-classical’ as defined previously (3). ‘UVX-REF’ include all the proteins identified in three representative RIC experiments (3,4,7). (D) Composition of the proteins with or without RNA interacting region defined in previous RBDmap based studies (2,5,6).
Figure 3. FAX-RIC enable more comprehensive profiling of RBPs through the enhanced capture efficiency. (A) Overlap between the UVX- and FAX-RIC based high confidence RNA interactome profiles. (B, C) Scatter plot of average LFQ intensity between UVX- and FAX-RIC experiments, drawn for RBPs, common to UVX and FAX RNA interactome (B) and exclusive to FAX RNA interactome (C). (D) Number of proteins with significantly greater LFQ intensity (>1 log2 fold-change) in UVX- or FAX-RIC, annotated with indicated classical and non-classical RBDs. (E) Relative LFQ intensities from the UVX- and FAX-RIC for the representative RBPs. Error bars represent mean standard error from three independent experiments. (F, G) Average LFQ intensity fold-change (FAX/UVX) for the RBPs, annotated with either ‘classical’ or ‘non-classical RBDs’ (F) and not annotated with such well characterized RBDs (G). RBPs were grouped by the identification frequency in total of nine UVX based RBP profiling studies (2,5,6).
Figure 4. FAX-RIC enable both comprehensive and unbiased RNA interactome profiling in Xenopus laevis oocyte and embryo. (A, B) Defining the high confidence UVX and FAX RNA interactome in X. laevis oocyte (stage VI) (A) and embryo (stage 8–9) (B). Volcano plots displaying the log2 fold-change of average LFQ intensity (x-axis) and the –log10 Student's t-test P value (y-axis) for all the proteins quantified in at least two out of three replicate UVX- or FAX-RIC experiments. Proteins with log2 fold-change >1 and statistically significant enrichment over the NoX-RIC experiments (P value <0.01, Student's t-test, adjusted by Benjamini–Hochberg method) are highlighted in red. (C) Proportion and number of human orthologous proteins in X. laevis RNA interactome that are annotated with GO:’RNA-binding’, previously defined (42) ‘RNA-related GO’ and the ‘RBD’, or identified as RBPs in previous RBP profiling studies (2,5,6) RBPome. (D, E) Overlap between UVX-RIC and FAX-RIC RNA interactome in oocyte (D) and embryo (E). (F) Number of the X. laevis oocyte nucleus enriched proteins, defined by having >0.5 protein amount ratio in X. laevis oocyte nucleus compared to the cytoplasm (31), identified in two or more replicate UVX- and FAX-RIC experiments in oocyte or embryo.
Figure 5. Transformation of mRNP complex landscape in X. laevis oocyte-to-embryo transition (OET). (A) Defining the differentially captured RBPs in oocyte or embryo stage FAX-RIC. Volcano plot displaying the log2 fold-change of average LFQ intensity and the –log10 Student's t-test P value (y-axis) for all the proteins identified as FAX RNA interactome in X. laevis are shown as black dots. Proteins with log2 fold-change >1.5 and had statistically significant enrichment in oocyte or embryo FAX-RIC experiments (P value <0.05, Student's t-test, adjusted by Benjamini–Hochberg method) are highlighted in red. (B) FAX-RIC enrichment level change in X. laevis OET and the respective change in total protein expression level. Scatter plot displaying the sum of the log2 protein expression level changes in oocyte maturation and early embryo development, previously reported by our group (55) (x-axis) and the respective change in average FAX-RIC LFQ intensity level (y-axis). All the identified proteins were shown by grey dots. Proteins whose FAX-RIC captured protein amount change can be explained by their respective change in total protein abundance during OET are highlighted in red. The ‘dynamic RBPs’, whose FAX-RIC enriched protein amounts are significantly changed while their respective total protein abundance were changed with log2 fold-change <0.5 are highlighted in light blue. (C) Scatter plot displaying average LFQ intensity from the FAX-RIC experiments in oocyte and embryo FAX-RIC (x-axis) and the change in FAX-RIC enrichment level between oocyte and embryo FAX-RIC (y-axis). RBPs are marked with light blue or red, as described in (B). The protein names are inserted for the targets with most significant change and/or enrichment level in FAX-RIC, along with the Tdrkh whose human homologue had similar change during the maturation of human oocyte (46). (D) Most significantly enriched biological process GO terms in the ‘dynamic RBPs’, as defined in (B). (E) Volcano plot same as (A) but the ‘dynamic RBPs’ are highlighted with light blue and the dynamic RBPs annotated with ‘GO: translation initiation’ are highlighted with red. The protein Eif4e is highlighted for its unexpected change. (F) Same as (E) but RBPs annotated with the ‘UniProt keyword: mRNA processing’ are highlighted with red.