Biology subjects

Grimme, A. L.

Publications and source records attributed to Grimme, A. L..

3 recordsLinked to original sources

bioRxiv2024

The RNA-binding activity of the TRIM-NHL protein NHL-2 is essential for miRNA-mediated gene regulation

The conserved TRIM-NHL protein, NHL-2, plays a key role in small RNA pathways in Caenorhabditis elegans. NHL-2 has been shown to interact with U-rich RNA through its NHL domain, but the importance to its biological function is unknown. We defined the crystal structure of the NHL domain to 1.4 [A] resolution and identified residues that affect affinity for U-rich RNA. Functional analysis of an NHL-2 RNA-binding loss-of-function mutant demonstrated defects in the heterochronic pathway, suggesting that RNA binding is essential for its role in this miRNA pathway. Processing bodies were enlarged in the NHL-2 RNA-binding mutant, suggesting a defect in mRNA decay. We also identified the eIF4E binding protein IFET-1 as a strong synthetic interactor with NHL-2 and the DEAD box RNA helicase CGH-1 (DDX6), linking NHL-2 function to translation repression. We demonstrated that in the absence of NHL-2, there was an enrichment of miRNA transcripts associated with the miRNA pathway Argonaute proteins ALG-2 and ALG-2. We demonstrate that NHL-2 RNA-binding activity is essential for let-7 family miRNA-mediated translational repression. We conclude that the NHL-2, CGH-1, and IFET-1 regulatory axes work with the core miRISC components to form an effector complex that is required for some, but not all, miRNAs.

genetics↗

bioRxiv2023

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited "closed" state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active "open" state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

molecular biology↗

bioRxiv2023

The catalytic activity of microRNA Argonautes plays a modest role in microRNA star strand cleavage in C. elegans

Many Argonaute proteins can cleave RNA ("slicing") as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use genome editing in C. elegans to examine the role of miRNA-guided slicing in organismal development. In contrast to previous work, slicing-inactivating mutations did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the absence of slicing, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on slicing for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on slicing. Gene expression changes were consistent with negligible to moderate loss of function for miRNA guides whose star strand was upregulated, suggesting a reduced proportion of mature miRISC in slicing mutants. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, a factor in the Target-Directed miRNA Degradation (TDMD) pathway. Overall, this work defines a role for miRNA Argonaute slicing in star strand decay; future work should examine whether this role could have contributed to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

molecular biology↗