Nascent RNA Folding Research Articles

Co-transcriptional folding of nascent RNA in the presence of RNA-binding proteins

Co-transcriptional folding of nascent RNA in the presence of RNA-binding proteins

Genome-wide probing of eukaryotic nascent RNA structure elucidates cotranscriptional folding and its antimutagenic effect

The transcriptional intermediates of RNAs fold into secondary structures with multiple regulatory roles, yet the details of such cotranscriptional RNA folding are largely unresolved in eukaryotes. Here, we present eSPET-seq (Structural Probing of Elongating Transcripts in eukaryotes), a method to assess the cotranscriptional RNA folding in Saccharomyces cerevisiae. Our study reveals pervasive structural transitions during cotranscriptional folding and overall structural similarities between nascent and mature RNAs. Furthermore, a combined analysis with genome-wide R-loop and mutation rate approximations provides quantitative evidence for the antimutator effect of nascent RNA folding through competitive inhibition of the R-loops, known to facilitate transcription-associated mutagenesis. Taken together, we present an experimental evaluation of cotranscriptional folding in eukaryotes and demonstrate the antimutator effect of nascent RNA folding. These results suggest genome-wide coupling between the processing and transmission of genetic information through RNA folding.

Coupling Between Production of Ribosomal RNA and Maturation: Just at the Beginning.

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35S/47S in yeast/human) is achieved by up to hundreds of RNA polymerase I (Pol I) enzymes simultaneously transcribing a single rRNA gene. In this review, we present recent advances in understanding the coupling between rRNA production and nascent rRNA folding. Mapping of the distribution of Pol I along ribosomal DNA at nucleotide resolution, using either native elongating transcript sequencing (NET-Seq) or crosslinking and analysis of cDNAs (CRAC), revealed frequent Pol I pausing, and CRAC results revealed a direct coupling between pausing and nascent RNA folding. High density of Pol I per gene imposes topological constraints that establish a defined pattern of polymerase distribution along the gene, with a persistent spacing between transcribing enzymes. RNA folding during transcription directly acts as an anti-pausing mechanism, implying that proper folding of the nascent rRNA favors elongation in vivo. Defects in co-transcriptional folding of rRNA are likely to induce Pol I pausing. We propose that premature termination of transcription, at defined positions, can control rRNA production in vivo.

Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates

SummaryTranscription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5′ enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.

Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding

Ribosome assembly is an efficient, but complex and heterogeneous process during which ribosomal proteins assemble on the nascent ribosomal RNA (rRNA) during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. We have developed a single-molecule fluorescence-microscopy approach for simultaneous tracking of transcription, nascent RNA folding and the assembly of proteins on the nascent transcript (Duss et al., Nat Comm, 2018; Duss et al., in revisions). Here, we use this approach to follow assembly of the entire 3’domain of the bacterial small ribosomal subunit in real-time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions, and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein complex (RNP). Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.

Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5' bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.

Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding

Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.

Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding

Ribosome assembly is an efficient, but complex and heterogeneous process during which ribosomal proteins assemble on the nascent ribosomal RNA (rRNA) during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence-microscopy, we follow assembly of the entire 3’domain of the bacterial small ribosomal subunit in real-time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions, and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein complex (RNP). Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and proposes that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.

Nascent RNA folding mitigates transcription-associated mutagenesis.

Transcription is mutagenic, in part because the R-loop formed by the binding of the nascent RNA with its DNA template exposes the nontemplate DNA strand to mutagens and primes unscheduled error-prone DNA synthesis. We hypothesize that strong folding of nascent RNA weakens R-loops and hence decreases mutagenesis. By a yeast forward mutation assay, we show that strengthening RNA folding and reducing R-loop formation by synonymous changes in a reporter gene can lower mutation rate by >80%. This effect is diminished after the overexpression of the gene encoding RNase H1 that degrades the RNA in a DNA–RNA hybrid, indicating that the effect is R-loop-dependent. Analysis of genomic data of yeast mutation accumulation lines and human neutral polymorphisms confirms the generality of these findings. This mechanism for local protection of genome integrity is of special importance to highly expressed genes because of their frequent transcription and strong RNA folding, the latter also improves translational fidelity. As a result, strengthening RNA folding simultaneously curtails genotypic and phenotypic mutations.

RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement.

The rates of RNA synthesis and nascent RNA folding into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA exit channel can increase pausing by interacting with bacterial RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain is proposed to mediate some effects of nascent RNA structures. However, the connections among RNA structure formation, clamp movement, and catalytic activity remain uncertain. We assayed exit-channel structure formation in Escherichia coli RNAP together with disulfide crosslinks that favor closed or open clamp conformations and found that clamp position directly influences RNA structure formation and catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening and through interactions with the flap that slow translocation.