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Unique Features of mRNA Quality Control Pathway Uncovered in Ciliated Protozoa
Nonsense-mediated mRNA decay (NMD) is an important mRNA quality control pathway that ensures efficient degradation of transcript with premature termination codons (PTC). This pathway is the therapeutic target in many genetic diseases, such as amyotrophic lateral sclerosis (ALS) and cystic fibrosis, etc. Protozoa consist of many biological and biomedically important free-living and parasitic early-branching eukaryotes. Studies on them have led to numerous scientific breakthroughs, such as discovery of telomere function, catalytic RNAs, etc. However, the components and molecular mechanisms of NMD are largely unknown in protozoa.
Researchers from Dr. Miao lab at Institute of Hydrobiology, Chinese Academy of Sciences, made the first attempt to take the advantage of transcriptome sequencing and mass spectrometry-based proteomics to uncover the unique and conserved aspects of NMD pathway in ciliated protozoan Tetrahymena thermophila. By characterization of the functions and protein-protein interactions of deeply conserved NMD factors, researchers have revealed the requirement of conserved up-frame shift proteins (Upf1, Upf2 and Upf3) for the Tetrahymena NMD, and ruled out the involvement of exon-junction complex (EJC) in its NMD. In addition, a novel nuclease that widely conserved in protozoa was found to be an interacting partner of the Tetrahymena Upf1, and its nuclease activity is possibly essential for removal of many PTC-containing transcripts. Moreover, the transcriptome-wide analysis of PTC-containing transcripts suggested that the spliceosomal intron located downstream of a termination codon could trigger NMD, which implying splicing related factors maybe required for PTC recognition in Tetrahymena.
The investigation of the NMD pathway in an early-branching eukaryote can contribute to the understanding of the evolution of NMD pathway, meanwhile it can also contribute to applying highly publicized CRISPR/Cas9 technologies todisrupt the gene of interest by inducing nonsense mutations that will be degraded by NMD in T. thermophila, as well as many other biomedically important protozoa. The study was published in Nucleic Acids Research.
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| (A) The PTC-introducing alternatively spliced first exon of UBC9(TTHERM_00522720)is significantly upregulated in ΔUPF1a compared with WT (indicated by the counting bin E003). Levels of the short horizontal lines indicate the relative expression of each exon region in different samples; the purple box indicates an alternative exon with significant differential expression. (B) Accumulation of sequencing reads derived from the PTC-introducing alternative exon (marked by red arrow) of UBC9 can be observed in ΔUPF1a and both SMG6L mutants, but not in WT and ΔMAG1 strains. All y-axes are set to the same scale. (C) Model of the gene encoding full-length Ubc9 protein, showing PTC-containing (PTC+) and normal (FUNC) transcripts. The open reading frame of each transcript is shown in black; triangles indicate the locations of primers used for RT-PCR analyses. (D) RT-PCR analyses of PTC-containing transcripts in different cells. “PTC+/FUNC” indicates the band intensity of the PTC-containing transcript relative to the normal transcript; CHX100, cycloheximide (100 μg/mL) treated cells. (E) Gene function, protein interaction, and transcript structural analyses have shown that the Tetrahymena NMD pathway functions in an EJC-independent manner. The evolutionary conserved NMD factor Upf1a plays a central role in the NMD pathway: it serves as binding platform for Upf2 (and possibly Upf3) and recruits the protozoa-specific nuclease Smg6L to degrade PTC-containing transcripts. Although NMD-targeted transcripts are enriched with exon–exon junctions downstream of the termination codon, the EJC core component Mag1 is not required for NMD, and not all EJC homologs can interact with one another. Therefore, further investigations are needed to identify possible novel factor(s) involved in PTC identification. |
