Abstract
The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the ‘regulatory loop (involved in mRNA binding)’. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: ‘open (isoform_2; without insertion)’ and ‘closed (isoform_1; with insertion)’. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the ‘open conformation’. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA–UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein–mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.
Highlights
Aberrant, misfolded, and mislocalized proteins are dangerous to cell viability due to their toxicity, which can be the cause of multiple human diseases such as Parkinson’s and Alzheimer’s diseases, frontotemporal dementia, cystic fibrosis, and several others [1,2,3,4,5,6,7].Natural selection pressures have led to a cellular quality control pathway that prevents aberrant proteins at the ribosome or that senses the aberrations in the mRNA templates, resulting in their degradation
The UP-frameshift 1 (UPF1) isoform_2 is composed of only an 11 aa ‘regulatory loop’ which weakens the UPF1 affinity for mRNA. This insertion of 11 amino acids to the ‘regulatory loop’ can considerably increase the catalytic and/or ATPase activity and result in a higher affinity for mRNA [71]. Considering such influence of the ‘regulatory loop’ in the structural dynamics of UPF1 and its control over the catalytic and/or ATPase activity, we investigated both isoforms of UPF1 in the presence or absence of the poly(U) mRNA by applying the molecular dynamics simulation (MDS) technique
Applying the MDS approach, we identified the selectivity of amino acids from two different UPF1 isoforms with poly(U) mRNA
Summary
Aberrant, misfolded, and mislocalized proteins are dangerous to cell viability due to their toxicity, which can be the cause of multiple human diseases such as Parkinson’s and Alzheimer’s diseases, frontotemporal dementia, cystic fibrosis, and several others [1,2,3,4,5,6,7]. 1 (PAB1 or PABPC1; poly(A)-binding protein cytoplasmic 1) [45,57,58,61,62,63,64] This scheme is referred to as the ‘faux 30 -UTR’ model, according to which the translation termination at a normal stop codon is considered to be essentially different from translation termination at a PTC; the aberrant nature of premature termination activates the mRNA decay [31,44]. NMD pathway, recognition of PTC‐containing mRNA transcript requires UPF1, UPF2, and UPF3 proteins as the core was modeled considering the isoform_2 crystal structure as the template (pdb id: 2wjv) [67]. Deletion of UPF1 in yeast stabilizes the nonsense‐containing complex (DECID), the SMG1 protein phosphorylates UPF1 that inhibits further translation termination initiation at the mRNAs and enhances nonsense codon read‐through as well and inhibits degradation. Such novel perspectives from mRNA–protein-binding pairs identified in this work may contribute to understanding the selectivity of respective partners, along with advancing NMD-associated structural dynamics and kinetics
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