A non-canonical initiation site is required for efficient translation of the dendritically localized Shank1 mRNA

PLoS One. 2014 Feb 12;9(2):e88518. doi: 10.1371/journal.pone.0088518. eCollection 2014.

Abstract

Local protein synthesis in dendrites enables neurons to selectively change the protein complement of individual postsynaptic sites. Though it is generally assumed that this mechanism requires tight translational control of dendritically transported mRNAs, it is unclear how translation of dendritic mRNAs is regulated. We have analyzed here translational control elements of the dendritically localized mRNA coding for the postsynaptic scaffold protein Shank1. In its 5' region, the human Shank1 mRNA exhibits two alternative translation initiation sites (AUG⁺¹ and AUG⁺²¹⁴), three canonical upstream open reading frames (uORFs1-3) and a high GC content. In reporter assays, fragments of the 5'UTR with high GC content inhibit translation, suggesting a contribution of secondary structures. uORF3 is most relevant to translation control as it overlaps with the first in frame start codon (AUG⁺¹), directing translation initiation to the second in frame start codon (AUG⁺²¹⁴). Surprisingly, our analysis points to an additional uORF initiated at a non-canonical ACG start codon. Mutation of this start site leads to an almost complete loss of translation initiation at AUG⁺¹, demonstrating that this unconventional uORF is required for Shank1 synthesis. Our data identify a novel mechanism whereby initiation at a non-canonical site allows for translation of the main Shank1 ORF despite a highly structured 5'UTR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Codon, Initiator / metabolism
  • DNA Mutational Analysis
  • Dendrites / metabolism*
  • Gene Deletion
  • Humans
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Open Reading Frames
  • Protein Biosynthesis
  • RNA, Messenger / metabolism
  • Transcription Initiation Site

Substances

  • 5' Untranslated Regions
  • Codon, Initiator
  • Nerve Tissue Proteins
  • RNA, Messenger
  • SHANK1 protein, human
  • SHANK1 protein, mouse