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

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.