Comprehensive Profiling of Roquin Binding Preferences for RNA Stem-Loops

Abstract

The cellular levels of mRNAs are controlled post-transcriptionally by cis-regulatory elements located in the 3'-untranslated region. These linear or structured elements are recognized by RNA-binding proteins (RBPs) to modulate mRNA stability. The Roquin-1 and -2 proteins specifically recognize RNA stem-loop motifs, the trinucleotide loop-containing constitutive decay elements (CDEs) and the hexanucleotide loop-containing alternative decay elements (ADEs), with their unique ROQ domain to initiate mRNA degradation. However, the RNA-binding capacity of Roquin towards different classes of stem-loops has not been rigorously characterized, leaving its exact binding preferences unclear. Here, we map the RNA-binding preference of the ROQ domain at nucleotide resolution introducing sRBNS (structured RNA Bind-n-Seq), a customized RBNS workflow with pre-structured RNA libraries. We found a clear preference of Roquin towards specific loop sizes and extended the consensus motifs for CDEs and ADEs. The newly identified motifs are recognized with nanomolar affinity through the canonical RNA-ROQ interface. Using these new stem-loop variants as blueprints, we predicted novel Roquin target mRNAs and verified the expanded target space in cells. The study demonstrates the power of high-throughput assays including RNA structure formation for the systematic investigation of (structural) RNA-binding preferences to comprehensively identify mRNA targets and elucidate the biological function of RBPs.

Keywords: RNA interactome; RNA structures; RNA-binding proteins; RNA–protein interactions.

Figure 1

sRBNS recapitulates known CDE preferences. A) Example structure of the protein‐RNA complex of the Roquin ROQ domain with the CDE 1 stem‐loop of UCP3 (PDB: 6TQB ^[20] ). B) Principle of an sRBNS experiment for high‐throughput identification of protein interactions with RNA structures. C) Experimental design with two CDE stem‐loops of the 3’‐UTR of UCP3 and TNF with three random loop nucleotides as input library and 50 nM ROQ domain of Roquin‐1. D) Distribution of the different stems in the input library and the pulldown library. E and F) Boxplot of the enriched motifs in the trinucleotide loops of UCP3 (E) and TNF (F) stems, respectively. Shown is the enrichment (sRBNS E) against the input at 50 nM ROQ domain concentration. Pool size n=64. Values greater than three standard deviations above the mean are considered significantly enriched and highlighted in blue (z‐score >3).

Figure 2

Characterization of the loop size and nucleotide composition in CDE loops. A) Input pool with different random loop sizes (N_3‐5) on top of the CDE stems from the UCP3 and TNF 3’‐UTRs. B) Boxplots of the enriched loop‐motifs against the input library (shown in A) at 10, 50 and 500 nM ROQ domain concentration. Pool size n=1344. Values greater than three standard deviations above the mean are highlighted in blue (z‐score >3). Lowercase letters indicate bases that form Watson–Crick base pairs. C) Preferred nucleotide logo composed of all possible trinucleotide loops weighted by their enrichment at 500 nM coreROQ domain. D) Luciferase reporter assay in HEK293 cells with wild type and mutated loops of the tandem CDE motif of the UCP3 3’‐UTR. Firefly luciferase activity was normalized to Renilla luciferase as an internal transfection control. Values are normalized to an empty vector control. n=5. Data are presented as mean ± standard deviation. Statistical significance was calculated by Student's t‐test (two‐tailed, paired), (**) P‐value <0.01.

Figure 3

Characterization of the nucleotide composition in ADE loops. A) Input pool with different random loops N₆ on top of the ADE stem from the HOMEZ and ITCH 3’‐UTRs. B) Boxplots of the enriched loop‐motifs against the input library (shown in A) at 10, 50 and 500 nM ROQ domain concentration. Values greater than three standard deviations above the mean are highlighted in blue (z‐score >3). Pool size n=4096. C) Preferred nucleotide logo composed of all possible hexanucleotide loops weighted by their enrichment at 500 nM coreROQ domain.

Figure 4

Roquin interacts with HOMEZ loop variants in an ADE‐like fashion. A) Overlay of ¹H,¹⁵N‐HSQCs of apo coreROQ (black) in complex with wild type HOMEZ (5’‐GUUUUA‐3’) (red). B) CSP plot showing differences of HOMEZ wild type ADE with coreROQ compared to apo coreROQ (derived from overlay in panel A). C) Overlays of ¹H,¹⁵N‐HSQCs of apo coreROQ (black) in complex with the different ADE variants (colored). D) Zoom‐ins of ¹H,¹⁵N‐HSQC spectra shown in A and B) for 5’‐GUUUUA‐3’, 5’‐AUUUUA‐3’ and 5’‐GUUUAA‐3’ loop ADEs in complex with coreROQ. Trajectories of CSPs are indicated by arrows.

Figure 5

Confirmation of predicted new Roquin target mRNAs in cells. A and B) Verification of siRNA‐mediated Roquin knockdown in HEK293 cells. A) RT‐qPCR quantification of mRNA levels of RC3H1 and RC3H2. Values are normalized to the housekeeping gene RPLP0. n=4. B) Western blot of Roquin‐1 and Roquin‐2. n=4. C) RT‐qPCR quantification of mRNA levels with new ADE elements after siRNA‐mediated knockdown of Roquin‐1 and Roquin‐2 in HEK293 cells. Values are normalized to the housekeeping gene RPLP0. n=4. D) Luciferase reporter assay in HEK293 cells with wild type and mutated loops of the ID4 ADE variant. Firefly luciferase activity was normalized to Renilla luciferase as an internal transfection control. Values are normalized to an empty vector control. n=4. Data in A), C) and D) are presented as mean ± standard deviation. Statistical significance was calculated by Student's t‐test (two‐tailed, paired), (**) P‐value <0.01. (*) P‐value <0.05.