Biology subjects

Kerkhofs, K.

Publications and source records attributed to Kerkhofs, K..

4 recordsLinked to original sources

bioRxiv2026

RSV competes with the host for translational machinery without a host shutoff strategy

RNA viruses often enhance ribosome recruitment to their own mRNAs through non-canonical sequence elements or by degrading host mRNA. Respiratory syncytial virus (RSV) produces mRNAs with host-like features, including 5'-cap and poly(A) tail. Therefore, the virus lacks an obvious mechanism to preferentially protect its own mRNAs or recruit ribosomes. Furthermore, it remains unknown how RSV interacts with antiviral defense pathways that would reduce cap-dependent translation. Using spike-in normalized sequencing of total and ribosome-associated RNA, we found that RSV does not appear to evoke any host shutoff mechanisms to limit the expression of host genes. These findings show that RSV manages to make use of available ribosomes by competing effectively with host mRNAs and any translational shutoff mechanism would be detrimental. Consistent with this, we found that following activation of antiviral host pathways that reduce cap-dependent translation, translation of RSV mRNAs is decreased to the same extent as host mRNAs. Furthermore, we found that RSV infection does not trigger the dsRNA-activated kinase PKR (which initiates the ISR) and OAS (activates endonuclease RNase L) pathways. These data support a model in which RSV achieves viral protein production, not though inhibiting the host, but by successfully competing with host mRNAs and avoiding activation of antiviral pathways.

molecular biology↗

bioRxiv2024

Respiratory Syncytial Virus (RSV) optimizes the translational landscape during infection

Viral infection often triggers eukaryotic initiator factor 2 (eIF2) phosphorylation, leading to global 5-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation. ImportanceViruses strongly rely on the hosts translational machinery to produce viral proteins required for replication. However, it is unknown how viruses that do not globally inhibit cap-dependent translation compete with abundant host transcripts for ribosomes. In this study, we found that respiratory syncytial virus (RSV) infection results in redistribution of 80S monosomes into the polysomes. High-throughput sequencing of translating transcripts revealed that low translation efficiency transcripts become more efficient at ribosome recruitment which are virus-resembling AU-rich host transcripts. Finally, we also uncover that AU-rich RNA binding protein RSV-M2-1 interacts with polysomes through contacts to mRNA. These findings revealed that RSV optimizes the translational landscape rather than inhibiting host translation.

molecular biology↗

bioRxiv2024

A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB

RNA polymerase III (Pol III, POLR3) synthesizes tRNAs and other small non-coding RNAs. Human POLR3 pathogenic variants cause a range of developmental disorders, recapitulated in part by mouse models, yet some aspects of POLR3 deficiency have not been explored. We characterized a human POLR3B:c.1625A>G;p.(Asn542Ser) disease variant that was found to cause mis-splicing of POLR3B. Genome-edited POLR3B1625A>G HEK293 cells acquired the mis-splicing with decreases in multiple POLR3 subunits and TFIIIB, although display auto-upregulation of the Pol III termination-reinitiation subunit POLR3E. La protein was increased relative to its abundant pre-tRNA ligands which bind via their U(n)U-3-termini. Assays for cellular transcription revealed greater deficiencies for tRNA genes bearing terminators comprised of 4Ts than of [≥]5Ts. La-knockdown decreased Pol III ncRNA expression unlinked to RNA stability. Consistent with these effects, small-RNAseq showed that POLR3B1625A>Gand patient fibroblasts express more tRNA fragments (tRFs) derived from pre-tRNA 3-trailers (tRF-1) than from mature-tRFs, and higher levels of multiple miRNAs, relative to control cells. The data indicate that decreased levels of Pol III transcripts can lead to functional excess of La protein which reshapes small ncRNA profiles revealing new depth in the Pol III system. Finally, patient cell RNA analysis uncovered a strategy for tRF-1/tRF-3 as POLR3-deficiency biomarkers.

molecular biology↗

bioRxiv2022

Altered tRNA processing is linked to a distinct and unusual La protein in Tetrahymena thermophila

Nascent pre-tRNAs are transcribed by RNA polymerase III and immediately bound by La proteins on the UUU-3OH sequence, using a tandem arrangement of the La motif and an adjacent RNA recognition motif-1 (RRM1), resulting in protection from 3-exonucleases and promotion of pre-tRNA folding. The Tetrahymena thermophila protein Mlp1 has been classified as a genuine La protein, despite the predicted absence of the RRM1. We found that Mlp1 functions as a La protein through binding of pre-tRNAs and affecting processing in Tetrahymena thermophila and when expressed in fission yeast. However, unlike in other examined eukaryotes, depletion of Mlp1 results in 3-trailer stabilization. We also observed that 3-trailers in Tetrahymena thermophila are uniquely short relative to other examined eukaryotes, and that 5-leaders have evolved to disfavour pre-tRNA leader/trailer pairing. Our data indicate that this variant Mlp1 architecture is linked to an altered, novel mechanism of tRNA processing in Tetrahymena thermophila.

molecular biology↗