Isoform-specific RNA structure determination using Nano-DMS-MaP.

RNA structure determination is essential to understand how RNA carries out its diverse biological functions. In cells, RNA isoforms are readily expressed with partial variations within their sequences due, for example, to alternative splicing, heterogeneity in the transcription start site, RNA processing or differential termination/polyadenylation. Nanopore dimethyl sulfate mutational profiling (Nano-DMS-MaP) is a method for in situ isoform-specific RNA structure determination. Unlike similar methods that rely on short sequencing reads, Nano-DMS-MaP employs nanopore sequencing to resolve the structures of long and highly similar RNA molecules to reveal their previously hidden structural differences. This Protocol describes the development and applications of Nano-DMS-MaP and outlines the main considerations for designing and implementing a successful experiment: from bench to data analysis. In cell probing experiments can be carried out by an experienced molecular biologist in 3-4 d. Data analysis requires good knowledge of command line tools and Python scripts and requires a further 3-5 d.

Nano-DMS-MaP allows isoform-specific RNA structure determination.

Genome-wide measurements of RNA structure can be obtained using reagents that react with unpaired bases, leading to adducts that can be identified by mutational profiling on next-generation sequencing machines. One drawback of these experiments is that short sequencing reads can rarely be mapped to specific transcript isoforms. Consequently, information is acquired as a population average in regions that are shared between transcripts, thus blurring the underlying structural landscape. Here, we present nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP)-a method that exploits long-read sequencing to provide isoform-resolved structural information of highly similar RNA molecules. We demonstrate the value of Nano-DMS-MaP by resolving the complex structural landscape of human immunodeficiency virus-1 transcripts in infected cells. We show that unspliced and spliced transcripts have distinct structures at the packaging site within the common 5' untranslated region, likely explaining why spliced viral RNAs are excluded from viral particles. Thus, Nano-DMS-MaP is a straightforward method to resolve biologically important transcript-specific RNA structures that were previously hidden in short-read ensemble analyses.

Cis-mediated interactions of the SARS-CoV-2 frameshift RNA alter its conformations and affect function.

The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through -1 programmed ribosomal frameshifting (PRF). -1PRF in the 1a/1b gene is essential for efficient viral replication and transcription of the viral genome. -1PRF efficiency relies on the presence of conserved RNA elements within the FSE. One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5' and 3' immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.

Short- and long-range interactions in the HIV-1 5' UTR regulate genome dimerization and packaging.

RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5' untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5' UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS-SL1 interaction unexpectedly couples the PBS with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.

RNA Structures and Their Role in Selective Genome Packaging.

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

A dual role of the ribosome-bound chaperones RAC/Ssb in maintaining the fidelity of translation termination.

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.

Modification of Selenoprotein mRNAs by Cap Tri-methylation.

Several selenoprotein mRNAs undergo 5' cap maturation events whereby their classical monomethylated m7G cap becomes trimethylated (m32,2,7G) by the trimethylguanosine synthase 1 (Tgs1). Here, we describe immunoprecipitation methods for the detection of endogenous m32,2,7G-capped selenoprotein mRNAs from total cell extracts or after polysome fractionation of cytoplasmic extracts. We have also developed a method for the in vitro cap hypermethylation of selenoprotein mRNA transcripts using purified Tgs1 enzyme.

SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation.

Selenoprotein synthesis requires the co-translational recoding of a UGASec codon. This process involves an RNA structural element, called Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2). Several selenoprotein mRNAs undergo unusual cap hypermethylation by the trimethylguanosine synthase 1 (Tgs1), which is recruited by the ubiquitous Survival of MotoNeurons (SMN) protein. SMN, the protein involved in spinal muscular atrophy, is part of a chaperone complex that collaborates with the methylosome for RNP assembly. Here, we analyze the role of individual SMN and methylosome components in selenoprotein mRNP assembly and translation. We show that SBP2 interacts directly with four proteins of the SMN complex and the methylosome core proteins. Nevertheless, SBP2 is not a methylation substrate of the methylosome. We found that both SMN and methylosome complexes are required for efficient translation of the selenoprotein GPx1 in vivo. We establish that the steady-state level of several selenoprotein mRNAs, major regulators of oxidative stress damage in neurons, is specifically reduced in the spinal cord of SMN-deficient mice and that cap hypermethylation of GPx1 mRNA is affected. Altogether we identified a new function of the SMN complex and the methylosome in selenoprotein mRNP assembly and expression.

Hypermethylated-capped selenoprotein mRNAs in mammals.

Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5'-end m(7)G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5'-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.