DOI: 10.5061/dryad.cvdncjt32
title: Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

description: 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 multipass
 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.
description: Data collection: A549 cells
 were plated at 1.5 × 10<sup>6</sup> cells per 10 cm dish.
 Three 10cm dishes were transfected with NSC siRNA whereas three other
 dishes were transfected with either siP1_1, siP2_1, or siP2_4 siRNAs as
 described above. 48 h later cells were infected with DENV-2 (NGC strain)
 at MOI of 10 in a total volume of 10 ml, rocked every 15 min for 1 h and
 the infection was allowed to proceed for an additional 1.5 h. Cells were
 then flash-frozen in liquid nitrogen without cycloheximide (CHX)
 pretreatment and a cold lysis buffer containing CHX was used to lyse the
 cells on ice. The RIBOseq strategy was adapted from Ingolia and
 colleagues with a few modifications. After nuclease digestion, samples
 were run on a 15–50% sucrose gradient and the 80S ribosome fractions were
 collected. As described by Reid and colleagues, fractions were extracted
 using Trizol LS (Thermo Fisher Scientific), and rRNAs were removed using
 the Ribo-Zero gold rRNA removal kit (Illumina, San Diego, CA, USA)
 according to the manufacturer's protocol. The remaining RNA was
 treated with PNK and then size selected by 15% denaturing PAGE. For
 adapter ligation and library building, we used the NEBNext Small RNA
 Library Prep Set (Illumina). Data can be accessed at the gene expression
 omnibus repository: GSE133111.  
 Data processing: RNA-seq
 and footprinting reads were jointly mapped to the human transcriptome and
 DENV genome using the riboviz pipeline (22). Sequencing adapters were
 trimmed from reads using Cutadapt 1.10 using parameters ‐‐trim-n -O 1
 ‐‐minimum-length 5. Trimmed reads that aligned to human/mouse rRNA were
 removed using HISAT2 version 2.1.0. The remaining reads were mapped to a
 set of 19,192 principal transcripts for each gene in the APPRIS database
 using HISAT2. Only reads that mapped uniquely were used for all downstream
 analyses. For genes with multiple principal transcripts, the first one on
 the list was chosen. Codes for selecting these transcripts were obtained
 from the riboviz package (https://github.com/shahpr/riboviz). 
description: The datasets from this Dryad repository should be used in
 conjunction with dengue_rplp1p2.Rmd in the
 associated GitHub repository - Dengue_RPLP1-P2 This repository contains *bam files (generated using HISAT2 mapping to human transcriptome), *h5 files generated using RiboViz, and several *tsv files. The final directory structure should be: | - scripts<br> | - data<br> |  | - *bam<br> |  | - *h5<br> |  | - *tsv<br> |  | - *csv<br> |  | - *pos<br> |  | - *txt<br> |  | - *xls<br> |  | - *gz<br> | - plots File types: Input files for the dengue_rplp1p2.Rmd script in Dengue_RPLP1-P2 *bam files - RNA-seq and ribosome-footprinting files HISAT2 aligned to principal transcripts (human_appris_data.principal.tsv) in the transcriptome (human_dengue.gff3). *bai files - Index files for each of the bam files. above. *h5 files - Processed data from RiboViz package providing position and length specific read counts for each transcript. See more about this file format here - https://github.com/riboviz/riboviz/blob/master/docs/reference/hdf5-data.md). *xls file - An excel file with information about transmembrane domain regions of human proteins. dengue.gene_pos - Nucleotide positions of Dengue genes on the Dengue genome human_dengue.gff3 - Transcript annotation file with information about 5' UTR length, CDS length, and 3' UTR length. This was generated using the RiboViz package.  human.ribo.csv - List of ribosomal proteins in the human genome human_appris_data.principal.txt - Principal transcript annotations for the APPRIS database dengue_rplp1p2.Rmd - A single R markdown notebook with all the codes for analysis and visualization of data associated with ribosome-profiling in Dengue. This file is available in the associated GitHub repository - Dengue_RPLP1-P2 human_features.tsv - Sequence-based features of human transcripts Output files from the dengue_rplp1p2.Rmd script in Dengue_RPLP1-P2 all_counts.tsv -  Transcript specific read counts for each condition all_tpms.tsv -  Transcript specific TPM for each condition periodicity_3n.tsv - Periodicity data for ribosome profiling datasets polarity_data.tsv - Polarity data for ribosome footprints on human transcripts TPM.w.foldchange.tsv - DESeq2 and riborex fold-changes codon_id.tsv.gz - Position-specific codon identities for each human transcript codon_sp_ribodens.tsv.gz - Codon-specific ribosome densities for each human transcript