Biology subjects

Nelissen, F. H. T.

Publications and source records attributed to Nelissen, F. H. T..

3 recordsLinked to original sources

bioRxiv2024

Decoupled degradation and translation enables noise-modulation by poly(A)-tails

Poly(A)-tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates, but is decoupled from translation. Interestingly, an optimal tail length of [~]100 nucleotides displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability--a feature of endogenous mRNAs--impacts translation efficiency but not mRNA degradation rates. Stochastic modelling combined with single-cell tracking reveals that poly(A)-tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.

molecular biology↗

bioRxiv2021

ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus. Significance StatementO_LIThere is a significant discrepancy between the dynamics of in vitro nucleolus models and in vivo nucleoli - with the latter more gel-like. C_LIO_LIThe interplay between Mg2+ ions, ATP and the nucleolus components - specifically RNA - governs the dynamics, and ultimately the physical state, of nucleolus-like condensates. C_LIO_LIWe show that the nucleolus are dynamically adapting condensates, responding to local ATP concentrations through Mg2+-induced compaction of the RNA, and reversible relaxation when ATP binds Mg2+ again. Other condensates containing RNA probably respond in similar ways to Mg2+ and ATP. C_LI

biophysics↗

bioRxiv2019

Ramified Rolling Circle Amplification for efficient and flexible synthesis of nucleosomal DNA sequences

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that using ramified rolling circle amplification (RCA) milligram amounts of a genomic nucleosomal DNA fragment can be produced in a scalable, one-pot reaction overnight. The ramified RCA reaction is more efficient than the existing methods, is flexible in DNA sequence and shows a 10-fold increase in yield compared to PCR, rivalling the production using plasmids. We demonstrate the method by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

biochemistry↗