Assembly factors chaperone ribosomal RNA folding by isolating helical junctions that are prone to misfolding

While RNAs are known to misfold, the underlying molecular causes have been mainly studied in fragments of biologically relevant larger RNAs. As these small RNAs are dominated by secondary structures, misfolding of these secondary structures remains the most-explored cause for global RNA misfolding. Conversely, how RNA chaperones function in a biological context to promote native folding beyond duplex annealing remains unknown. Here, in a combination of dimethylsulfate mutational profiling with sequencing (DMS-MaPseq), structural analyses, biochemical experiments, and yeast genetics, we show that three-helix junctions are prone to misfolding during assembly of the small ribosomal subunit in vivo. We identify ubiquitous roles for ribosome assembly factors in chaperoning their folding by preventing the formation of premature tertiary interactions, which otherwise kinetically trap misfolded junctions, thereby blocking further progress in the assembly cascade. While these protein chaperones act indirectly by binding the interaction partners of junctions, our analyses also suggest direct roles for small nucleolar RNAs (snoRNAs) in binding and chaperoning helical junctions during transcription. While these assembly factors do not utilize energy to ameliorate misfolding, our data demonstrate how their dissociation renders reversible folding steps irreversible, thereby driving native folding and assembly and setting up a timer that dictates the propensity of misfolded intermediates to escape quality control. Finally, the data demonstrate that RNA chaperones act locally on individual tertiary interactions, in contrast to protein chaperones, which globally unfold misfolded proteins.

Molecular mechanisms underlying the extreme mechanical anisotropy of the flaviviral exoribonuclease-resistant RNAs (xrRNAs)

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using a single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg2+ and the key tertiary interactions. The absence of Mg2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structures in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations in combination with force distribution analysis. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.

Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

ABSTRACTDuring infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3′ untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA for destruction. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific 3-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5′ end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3′ UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3′ UTR of the Tamana Bat Virus (TABV) and solved its structure by x-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

Molecular mechanisms underlying the extreme mechanical anisotropy of the flaviviral exoribonuclease-resistant RNAs (xrRNAs)

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg2+ and the key tertiary interactions. The absence of Mg2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structure in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations on ZIKV xrRNA1 and another two RNAs with ring structures, the HCV IRES and THF riboswitch. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.

Time-resolved, single-molecule, correlated chemical probing of RNA

Methods for capturing the folding dynamics of functionally important RNAs, especially large RNAs, have relied primarily on global measurements of structure or on per-nucleotide chemical probing. These approaches infer, but do not directly measure, through-space tertiary interactions. Here we introduce trimethyloxonium (TMO) as a chemical probe for RNA. TMO enables time-resolved, single-molecule, through-space structure probing of RNA folding using a correlated chemical probing framework. TMO methylates RNA about 90 times faster than the widely used dimethyl sulfate probe, allowing structure interrogation on the second time scale. We used TMO to monitor folding of the RNase P RNA – a functional RNA with extensive long-range and noncanonical interactions – by direct measurement of through-space tertiary interactions in a time-resolved way. Time-dependent correlation changes directly revealed the central role of a long-range tertiary loop-loop interaction that guides native RNA folding. Single-molecule, time-resolved RNA structure probing with TMO is poised to reveal a wide range of dynamic RNA folding processes and principles of RNA folding.