Sequencing refractory regions in bird genomes are hotspots for accelerated protein evolution

Abstract

Background Approximately 1000 protein encoding genes common for vertebrates are still unannotated in avian genomes. Are these genes evolutionary lost or are they not yet found for technical reasons? Using genome landscapes as a tool to visualize large-scale regional effects of genome evolution, we reexamined this question. Results On basis of gene annotation in non-avian vertebrate genomes, we established a list of 15,135 common vertebrate genes. Of these, 1026 were not found in any of eight examined bird genomes. Visualizing regional genome effects by our sliding window approach showed that the majority of these "missing" genes can be clustered to 14 regions of the human reference genome. In these clusters, an additional 1517 genes (often gene fragments) were underrepresented in bird genomes. The clusters of “missing” genes coincided with regions of very high GC content, particularly in avian genomes, making them “hidden” because of incomplete sequencing. Moreover, proteins encoded by genes in these sequencing refractory regions showed signs of accelerated protein evolution. As a proof of principle for this idea we experimentally characterized the mRNA and protein products of four "hidden" bird genes that are crucial for energy homeostasis in skeletal muscle: ALDOA, ENO3, PYGM and SLC2A4. Conclusions A least part of the “missing” genes in bird genomes can be attributed to an artifact caused by the difficulty to sequence regions with extreme GC% (“hidden” genes). Biologically, these “hidden” genes are of interest as they encode proteins that evolve more rapidly than the genome wide average. Finally we show that four of these “hidden” genes encode key proteins for energy metabolism in flight muscle.

Chicken GLUT4 undergoes complex alternative splicing events and its expression in striated muscle changes dramatically during development

“…Finally, we identified at least 14 kinds of chicken GLUT4 isoforms through assembling the 5′- and 3′-RACE products. In this period, Huttener et al reported a series "missing" genes (including GLUT4, ALDOA, ENO3 , and PYGM ) in birds ( Huttener et al, 2021 ), and the chicken GLUT4 sequence they got was just the predicted ORF of T1 transcript, which shed a light on our work. In their study, they also mentioned their hard work for getting bird′s GLUT4 mRNA encoding the C-terminus.…”

“…It reflected the rapid evolution of GLUT4 genome in chicken. Huttener et al also mentioned chicken GLUT4 protein undergo rapid evolution across species ( Huttener et al, 2021 ). The rapid evolution of GLUT4 in chicken may contribute to the complex transcript feature and even the specific glucose metabolism feature in birds ( Li et al, 2021 ).…”

Genomic insights into metabolic flux in hummingbirds

“…Included in these novel genes are genes critical to metabolism, including ALDOA , PFKM , G6PD , PGLS , PC , PCK2 , PFKFB1 , PYGM , and PLPPR1 . Many of these genes (e.g., ALDOA and PYGM ) have been historically “missing” from avian assemblies owing to being “hidden” in GC-rich areas that fail to assemble with short-read sequencing ( Huttener et al 2021 ; Rhie et al 2021 ; Kim et al 2022 ). The combination of hybrid genome and transcriptome assembly and deep sequencing of multiple tissues in the fasted and fed condition enabled assembly and annotation of these genes.…”

Identification of GC-rich LAT genes in birds

“…The most probable reason, why LAT was not identified in Neognathae birds before, is the high content of GC-rich sequences. These represent the main obstacle in successful amplification and assembly of a subset of avian genes [ 18 , 23 – 25 ]. The potential evolutionary cause for this high GC content in avian LAT is not clear.…”