Jennifer Kothe (Giessen / DE), Theresa Dietz (Giessen / DE), Till Sauerwein (Köln / DE), Konrad U. Förstner (Köln / DE), Elena Evguenieva-Hackenberg (Giessen / DE)
Introduction: Small genes can have important function but are often poorly annotated. Recently we applied ribosome profiling (Ribo-seq) and mass spectrometry to detect translated sORFs in the soil-dwelling plant symbiont Sinorhizobium meliloti 2011 and to curate its annotation.
Goals: Two of the translated sORFs were further analyzed in order to understand their function.
Materials & Methods: We used reporter fusions, applied mutagenesis, and performed RNA-Seq, Northern blot, qRT-PCR, and Western blot analyses.
Results: The first analyzed small gene is a 29 aa novel sORF (sORF26) located upstream of a DUF1127 gene; the latter showed strongest induction after exposure to tetracycline. We found that inhibition of sORF26 translation is responsible for the induction of the DUF1127 gene. Distinct elements in sORF26 and in the intergenic region that are necessary for this posttranscriptional regulation were identified. The second analyzed small gene is a rpsU paralog (rpsU2), which was missed in the original genome annotation. Under standard laboratory conditions, expression of rpsU2 was stronger than that of the originally annotated rpsU1. Most recent data on the suggested ribosome heterogeneity in S. meliloti will be shown.
Summary: The novel sORF26 is a regulatory upstream ORF, translation of which is needed for repression of the downstream DUF1127 gene under standard conditions and strong induction upon exposure to translation inhibiting antibiotics. Existence and differential expression of two S21 genes in S. meliloti suggests ribosome heterogeneity, which is under investigation.
References:
Hadera"s L, Heiniger B, Maaß S, Scheuer R, Gelhausen R, Azarderakhsh S, Barth-Weber S, Backofen R, Becher D, Ahrens CH, Sharma CM, Evguenieva-Hackenberg E. (2023) Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics. Microlife 10;4:uqad012. doi: 10.1093/femsml/uqad012