DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells

Maya David, Tsviya Olender, Orel Mizrahi, Shira Weingarten-Gabbay, Gilgi Friedlander, Sara Meril, Nadav Goldberg, Alon Savidor, Yishai Levin, Vered Salomon, Noam Stern-Ginossar, Shani Bialik, Adi Kimchi

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Death associated protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate noncanonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knockdown (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing. A cohort of 68 genes showed decreased translation efficiency in DAP5 KD cells. Mass spectrometry confirmed decreased protein abundance of a significant portion of these targets. Among these was KMT2D, a histone methylase previously shown to be essential for ESC differentiation and embryonic development. We found that nearly half of the cohort of DAP5 target mRNAs displaying reduced translation efficiency of their main coding sequences upon DAP5 KD contained upstream open reading frames (uORFs) that are actively translated independently of DAP5. This is consistent with previously suggested mechanisms by which DAP5 mediates leaky scanning through uORFs and/or reinitiation at the main coding sequence. Crosslinking protein–RNA immunoprecipitation experiments indicated that a significant subset of DAP5 mRNA targets bound DAP5, indicating that direct binding between DAP5 protein and its target mRNAs is a frequent but not absolute requirement for DAP5-dependent translation of the main coding sequence. Thus, we have extended DAP5’s function in translation of specific mRNAs in hESCs by a mechanism allowing translation of the main coding sequence following upstream translation of short ORFs.

Original languageEnglish
Pages (from-to)1325-1336
Number of pages12
JournalRNA
Volume28
Issue number10
DOIs
StatePublished - Oct 2022
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2022 David et al.

Keywords

  • DAP5
  • RNA-seq
  • noncanonical protein translation
  • pluripotent embryonic stem cells
  • ribosome profiling
  • uORF

Fingerprint

Dive into the research topics of 'DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells'. Together they form a unique fingerprint.

Cite this