Tisne, C. | bioRxiv Science

bioRxiv2019

Time-resolved NMR monitoring of tRNA maturation

Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect the introduction of these modifications during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. Here we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNAPhe with time-resolved NMR measurements, we found that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we uncovered a strong hierarchy in the introduction of the T54, {Psi}55 and m1A58 modifications in the T-arm, and demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.

molecular biology↗

bioRxiv2019

The HIV-1 ribonucleoprotein dynamically regulates its condensate behavior and drives acceleration of protease activity through membrane-less granular phase-separation

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase separated molecular sponges, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ~2400 Gag and ~120 GagPol by viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

molecular biology↗