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The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98 nt sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three or two stem-loop structures that result from the different base-pairing interactions within the first 55 nt. Here, we used single-molecule Förster resonance energy transfer spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes.

Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α-primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and in human cells they cause telomere shortening that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their binding to GQ RNA and their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-₄₁AACAAU₄₆ region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.

Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here, we use biophysical assays to interrogate the k _on, k _off, and K _d for DNA and RNA binding of two model human TFs, ERα and Sox2. Unexpectedly, we found that both proteins exhibit multiphasic nucleic acid-binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics can be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ERα nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, in which two apparent binding transitions are separated by a 10-20 min "lag" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ERα nucleic acid-binding data. Instead, simulations with a model incorporating sequential ERα monomer association, ERα nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ERα DNA and RNA binding. Collectively, our findings reveal that Sox2 and ERα bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ERα binding nucleic acids via a novel reaction mechanism.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg²⁺). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg²⁺ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 μsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg²⁺ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg²⁺ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg²⁺ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg²⁺ and ligand also facilitate a more compact structure in the three-way junction region.

The long noncoding RNA TERRA is transcribed from telomeres in virtually all eukaryotes with linear chromosomes. In humans, TERRA transcription is driven in part by promoters comprising CpG dinucleotide-rich repeats of 29 bp repeats, believed to be present in half of the subtelomeres. Thus far, TERRA expression has been analyzed mainly using molecular biology-based approaches that only generate partial and somehow biased results. Here, we present a novel experimental pipeline to study human TERRA based on long-read sequencing (TERRA ONTseq). By applying TERRA ONTseq to different cell lines, we show that the vast majority of human telomeres produce TERRA and that the cellular levels of TERRA transcripts vary according to their chromosomes of origin. Using TERRA ONTseq, we also identified regions containing TERRA transcription start sites (TSSs) in more than half of human subtelomeres. TERRA TSS regions are generally found immediately downstream from 29 bp repeat-related sequences, which appear to be more widespread than previously estimated. Finally, we isolated a novel TERRA promoter from the highly expressed subtelomere of the long arm of Chromosome 7. With the development of TERRA ONTseq, we provide a refined picture of human TERRA biogenesis and expression and we equip the scientific community with an invaluable tool for future studies.