Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

Abstract

The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.

Figure 1.

Ribosome profiling of DENV-infected control and RPLP1/2-depleted cells. (A) A549 cells were transfected with either non-silencing control (NSC) or a pool of siRNAs (siP). After 48 h, cells were infected with DENV at MOI of 10 at the indicated times points (hours) for western blot analysis of NS3 and β-actin. The right panel shows normalized NS3 levels for NSC (blue) and siP (red) transfected cells. (B) Infected cells that were transfected with the indicated siRNAs were fractionated into ER and cytosol compartments. Western blot of the ER resident EMC4 and cytosolic protein GAPDH is shown. RT-qPCR was used to quantify DENV RNA in each compartment under the different conditions. (C) Experimental design of the ribosome profiling experiment. (D) Ribosome density (RPF normalized to RNAseq) on the DENV RNA under control and RPLP1/2 knockdown conditions is shown (***P < 0.001). Normalized RIBOseq reads on the DENV RNA for one representative sample of NSC (E) and siP (F) are shown. All graphs are shown in Supplementary Figure S6.

Figure 2.

Effects of RPLP1/2 knockdown on expression of DENV structural proteins. (A) Schematic of DENV structural proteins and their topology in the ER membrane is shown. (B–E) Tetracycline inducible HeLa cells lines expressing the indicated tagged proteins were depleted for RPLP1/2 and analyzed by quantitative western blotting using anti-HA, anti-FLAG or anti-E antibodies. Representative experiments are shown and their quantification is shown as graphs on the right which represent mean values ± SD. Asterisks represent P values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.

Deletion of transmembrane domain (TMDs) within the prM protein abrogates the effect of RPLP1/2 knockdown on viral protein expression. (A) Tetracycline inducible HeLa cells expressing DENV structural proteins were transfected with control or RPLP1/2 siRNAs and levels of HA-C-prM and EΔ208-FLAG were analyzed by western blot using the respective tag antibodies. Analysis of cells expressing variants lacking TMD3 (B) or both TMD 2 and 3 (C) is shown. Representative experiments and shown and their quantifications are shown on the right. The graphs show mean values ± SD. Asterisks indicate p values: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4.

RPLP1/2 knockdown increases the rates of DENV structural protein synthesis and turnover. HeLa cells expressing HA-C-prM-EΔ208-FLAG were transfected with control or RPLP1/2 siRNAs and then metabolically pulse-labeled with ³⁵S-methionine/cysteine for 30 min followed by chase with cold methionine/cysteine. Cells were harvested immediately after the pulse, 3 or 6 hours after the chase for lysis and IP with a-HA and a-FLAG antibodies. (A) An autoradiogram of triplicate IP samples for HA-C-prM and EΔ208-FLAG is shown for the pulse (0 h) and chase samples. Controls in the two leftmost lanes were not induced with tetracycline. (B) The left panel shows raw quantification of band intensities and the right panel shows levels normalized to the 0 h time point for HA-C-prM. (C) Same as in (B) except data show EΔ208-FLG. A representative experimenti is shown and its quantifications were done by averaging the measurements of triplicate bands in the autoradiagram.

Figure 5.

Effects of RPLP1/2 depletion on transcript levels and ribosome density for cellular mRNAs. The scatter plot of (A) RNAseq and (B) RIBOseq abundances between non-silencing control (NSC) and a pool of siRNAs (siP) datasets. Estimates of abundances are based on average TPMs from triplicates. Red dots are genes identified at significantly different based on DESeq2 analysis [q-value < 0.01, abs(log₂ fold change) > 1]. (C) Comparison between RNA and RPF fold-changes indicate that most RPF changes are driven by changes at the RNA level. In total, 19 192 genes were analyzed, with 196 having changes only in translation, 25 having changes only in transcripts (translation buffering), 78 having changes in both and 18 893 remaining unchanged.

Figure 6.

Effects of RPLP1/2 knockdown on translation of mRNAs encoding proteins with multiple TMDs. (A) Metagene analysis of the average ribosome density from all expressed mRNAs is shown. Data were aligned at the start codon (position 0) for control (NSC, blue line) or RPLP1/2 knock down (siP, red line). RPLP1/2 depletion caused an accumulation of ribosomes in the first 100 codons. (B) Effects of RPLP1/2 knockdown on transcript levels (RNAseq) or RPFs (RIBOseq) encoding proteins with the indicated number of TMDs. (C) RNAseq and RIBOseq of proteins encoding the indicated number of TMDs shown as overlapping distributions of log₂ fold change.

Figure 7.

RPLP1/2 depletion leads to ribosome pausing after TMDs on cellular mRNAs. (A–F) Examples of cellular genes encoding multiple TMDs which show an increase in the ribosome pausing downstream of the TMDs when RPLP1/2 are depleted.