HIV-1 Packaging Visualised by In-Gel SHAPE

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA–protein complex. Here, we apply the in-gel SHAPE (selective 2’OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag–dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP’s association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5′ end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5′ end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

Interbase FRET in RNA: from A to Z

Interbase FRET can reveal highly detailed information about distance, orientation and dynamics in nucleic acids, complementing the existing structure and dynamics techniques. We here report the first RNA base analogue FRET pair, consisting of the donor tCO and the non-emissive acceptor tCnitro. The acceptor ribonucleoside is here synthesised and incorporated into RNA for the first time. This FRET pair accurately reports the average structure of A-form RNA, and its utility for probing RNA structural changes is demonstrated by monitoring the transition from A- to Z-form RNA. Finally, the measured FRET data were compared with theoretical FRET patterns obtained from two previously reported Z-RNA PDB structures, to shed new light on this elusive RNA conformation.

RSQ: a statistical method for quantification of isoform-specific structurome using transcriptome-wide structural profiling data

The structure of RNA, which is considered to be a second layer of information alongside the genetic code, provides fundamental insights into the cellular function of both coding and non-coding RNAs. Several high-throughput technologies have been developed to profile transcriptome-wide RNA structures, i.e., the structurome. However, it is challenging to interpret the profiling data because the observed data represent an average over different RNA conformations and isoforms with different abundance. To address this challenge, we developed an RNA structurome quantification method (RSQ) to statistically model the distribution of reads over both isoforms and RNA conformations, and thus provide accurate quantification of the isoform-specific structurome. The quantified RNA structurome enables the comparison of isoform-specific conformations between different conditions, the exploration of RNA conformation variation affected by single nucleotide polymorphism (SNP),and the measurement of RNA accessibility for binding of either small RNAs in RNAi-based assays or RNA binding protein in transcriptional regulation. The model used in our method sheds new light on the potential impact of the RNA structurome on gene regulation.