RNA最新文献

Circular RNA (circRNA) is emerging as a highly promising technology in various biomedical applications, offering advantages over traditional linear RNA. The Twister-optimized RNA for the durable overexpression (Tornado) system has been widely investigated for generating circRNAs in mammalian cells; however, the use of the Tornado system for large RNA inserts, especially those containing the internal ribosome entry site (IRES) sequences, is hindered by low circularization efficiency and limited circRNA abundance. Therefore, developing novel strategies to enhance RNA circularization in cells is of critical importance. In this study, we present a modified Tornado system that significantly improves circRNA-based protein expression by incorporating an optimal distance between the IRES and the upstream CMV promoter. Furthermore, we elucidate the dual roles of HRV-B3 IRES in mammalian cells, demonstrating its negative regulatory effect on RNA abundance and its positive contribution to RNA circularization. Additionally, the integration of a truncated 5' long terminal repeat (LTR) from HIV-1 upstream of the HRV-B3 IRES, combined with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), further enhances transcriptional efficiency in the Tornado system. This modified system holds great potential for advancing circRNA-based therapeutics and vaccines, and these findings provide valuable insights for refining the Tornado system and designing regulatory elements in synthetic biology applications.

Adenosine deaminase acting on RNA 1 (ADAR1) edits double-stranded RNA (dsRNA) substrates by the deamination of adenosine to inosine in a process known as A-to-I editing. Modulation of ADAR1 expression and editing activity has previously been described to play a role in cancer development and progression, with upregulation of ADAR1 being observed in a range of cancers. Further, depletion of ADAR1 leads to increased sensing of endogenous dsRNAs by dsRNA sensors in cell lines that require ADAR1 for survival, which are termed ADAR1-dependent. The activation of these sensors induces downstream production of type I interferons as well as translational inhibition and apoptosis. Therefore, ADAR1 is a promising oncologic therapeutic target. Recently, the small molecule ZYS-1 has been developed and presented as a direct inhibitor of ADAR1. We performed a series of in vitro and cellular experiments to validate the efficacy and specificity of ZYS-1 as an ADAR1 inhibitor. Evaluating the effect of ZYS-1 on cell viability revealed it to be equally cytotoxic to both ADAR1-dependent and ADAR1-independent cell lines, as well as wild-type and ADAR1 knockout cells. Moreover, ZYS-1 treatment had little effect on activation of PKR or induction of IFN stimulated genes. Importantly, treatment with ZYS-1 did not reduce cellular A-to-I editing for several known ADAR1 editing sites and did not inhibit in vitro A-to-I editing by recombinant ADAR1. Together, these data indicate that ZYS-1 is not a selective inhibitor of ADAR1.

The ATP-dependent RNA helicase Up-frameshift 1 (UPF1) is an essential protein in mammalian cells and a key factor in nonsense-mediated mRNA decay (NMD), a translation-dependent mRNA surveillance process. UPF1 is mainly cytoplasmic at steady state but accumulates in the nucleus after inhibiting CRM1-mediated nuclear export by leptomycin B (LMB), indicating that UPF1 shuttles between the nucleus and the cytoplasm. Consistent with its dual localization, there is evidence for nuclear functions of UPF1, for instance, in DNA replication, DNA damage response, and telomere maintenance. However, whether any of UPF1's biochemical activities are required for its nuclear-cytoplasmic shuttling remains unclear. To investigate this, we examined two UPF1 mutants: the well-described ATPase-deficient UPF1-DE (D636A/E637A) and a newly generated RNA-binding mutant UPF1-NKR (N524A/K547A/R843A). Biochemical assays confirmed that the UPF1-NKR mutant cannot bind RNA or hydrolyze ATP in vitro but retains interaction with UPF2, UPF3B, and SMG6. Overexpression of UPF1-NKR exerted a dominant-negative effect on endogenous UPF1 and inhibited NMD. Subcellular localization studies revealed that UPF1-DE accumulates in cytoplasmic granules (P-bodies), even in the presence of LMB, whereas UPF1-NKR shuttles normally. This indicates that UPF1's shuttling does not require its RNA-binding or ATPase activities. Notably, the UPF1-DE.NKR double mutant restored nuclear-cytoplasmic shuttling and prevented accumulation in P-bodies, suggesting that the shuttling defect of UPF1-DE arises from its tight binding to RNA. Overall, our findings demonstrate that UPF1's shuttling is independent of its ATPase and RNA-binding activities, with RNA binding itself being a key determinant of its cytoplasmic retention.

Local protein synthesis in neurons is vital for synaptic maintenance and plasticity, yet the regulatory mechanisms, particularly cytoplasmic polyadenylation, are not fully understood. This study used nanopore sequencing to examine transcriptomic responses and 3'-end dynamics in rat hippocampal long-term potentiation (LTP) in vivo and in synaptoneurosomes after in vitro stimulation. Our long-read transcriptomic data set allows for detailed analysis of mRNA 3'-ends, poly(A) tail lengths, and nucleotide composition. We observed dynamic shifts in polyadenylation site preference post-LTP induction, with significant poly(A) tail lengthening restricted to transcriptionally induced mRNAs. The poly(A) tails of these genes showed increased nonadenosine abundance. In synaptoneurosomes, chemical stimulation led to the shortening of poly(A) tails on preexisting mRNAs, indicating translation-induced deadenylation. This also includes transcripts, which were previously reported to undergo stimulation-induced cytoplasmic polyadenylation, like Camk2a Additionally, we discovered a group of neuronal transcripts with poly(A) tails abundant in nonadenosine residues. These tails are semi-templated and derived from extremely adenosine-rich 3'UTRs. This study provides a comprehensive overview of mRNA 3'-end dynamics during LTP, offering insights into post-transcriptional regulation following synaptic activation of plasticity in neurons.

The stem-loop-II motif (s2m) is a conserved viral RNA element located in the 3'UTR of different viruses including SARS-CoV-2. High-resolution 3D structural data for s2m are only available for the fundamentally different SCoV-1 version and difficult to access for SARS-CoV-2 due to the highly dynamic nature of the s2m RNA element. With the Omicron variant, a large deletion occurred for s2m, resulting in a relatively short hairpin with an apical pentaloop. We determined the NMR solution structure of s2m_omicron using a variety of torsion-angle sensitive NMR parameters in addition to NOE distance restraints. Surprisingly, relatively high {¹H},¹³C heteronuclear NOE values, averaged ribose ³J_HH-coupling constants (H1'H2'; H3'H4'), and dipole(H1'-C1'),-dipole(H6/8-C6/8)-CCRs hinted toward significant dynamics for the small pentaloop making structure calculations solely relying on NMR data insufficient. To address this problem, we performed ten 1 microsecond MD-simulations from the NMR structure bundle as a starting point and applied Bayesian maximum entropy (BME) reweighting to refine the ensemble with the ³J-coupling constant data. Our results from the combined methodology provide a detailed view of the conformational dynamics of the Omicron variant of s2m characterized by different stacking patterns, ribose repuckering, and overall heterogeneity of the torsion angles for the loop nucleotides. Strikingly, despite the deletion of the initial nonaloop, as present in the Wuhan and Delta variants of s2m, our combined methodology reveals substantial dynamics and reorganization of a conserved UAC triplet at the tip of the pentaloop, adding physical insight that may be leveraged for the ultimate determination of the still unknown function of the RNA element.

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO₄ via a two-step mechanism in which (i) the 2'-PO₄ attacks NAD⁺ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. We showed previously that 2″OMeNAD⁺, a synthetic NAD⁺ analog that cannot support step 2 transesterification, is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate as an abortive product. Here we report the chemical synthesis of two novel 2″OMeNAD⁺ compounds, containing desthiobiotin (DTB) linked to adenine C2 or N6 via a diaminohexane linker. Whereas both analogs poison the RslTpt1 reaction after step 1, the 2″OMeNAD-2-DTB derivative supports a higher yield of RNA-2'-phospho-(DTB-ADP-2″OMe-ribose) product, which can be recovered by adsorption to streptavidin beads and elution with biotin. Our results recommend 2″OMeNAD-2-DTB as a novel affinity-tag probe of RNA 2'-phosphate modification. We synthesized a fluorescent derivative, 2″OMeNAD-2-TAMRA, and found that it, too, is an effective step 1 substrate for RslTpt1 that allows fluorescent labeling of an RNA 2'-phosphate.

One of the striking characteristics of eukaryotic genomes is the presence of three types of introns: spliceosomal introns, tRNA introns, and a unique intron in the XBP1 mRNA. Exceptional eukaryotic genomes that lack spliceosomal or XBP1 introns have been described. However, tRNA introns and the tRNA endonuclease that is required for their splicing are thought to be universal in eukaryotes. The introns in three tRNAs are widely conserved across Metazoa: Tyr-GUA, Ile-UAU, and Leu-CAA. This study shows that some nematode species have lost the introns in Tyr-GUA and Ile-UAU tRNAs, and one species, Levipalatum texanum, completely lacks tRNA introns. The loss of the intron from Leu-CAA tRNA is accompanied by an unusual A-C mismatched base pair in the anticodon stem-loop and a triplication of a tRNA deaminase that could potentially restore base-pairing. These changes may be an adaptation to the loss of the intron. L. texanum also lacks the tRNA endonuclease, one of two enzymes required for tRNA splicing. The other key enzyme in tRNA splicing, tRNA ligase, is bifunctional and is also required for XBP1 mRNA splicing. L. texanum retains tRNA ligase and the XBP1 intron. This eukaryote without tRNA introns has the potential to be a valuable tool for disentangling the functions of tRNA splicing, XBP1 splicing, and tRNA modification enzymes and is the only animal known to have lost one of the three intron types.

RNA G-quadruplexes (rG4s) are unusual RNA secondary structures formed by stacking arrays of guanine tetrads. Although thousands of potential rG4-forming motifs occur throughout the mammalian transcriptome, many single-stranded RNA (ssRNA) viruses are thought to be depleted of rG4-forming sequences. Using in silico methods, we examine rG4-forming potential in single-stranded RNA (ssRNA) viruses and observe that, while canonical rG4 motifs are depleted, noncanonical rG4 motifs occur at comparable or higher frequencies relative to the mammalian transcriptome. We ask if the noncanonical rG4's can be leveraged to block viral replication and control infection using OC43, the coronavirus believed to be responsible for the 1889 "Russian flu" pandemic. Profiling with "d-rG4-seq" confirms a dearth of folded rG4 in the OC43 RNA genome during natural infection. Intriguingly, rG4 ligands induce synthetic rG4 structures of a noncanonical nature. Significantly, induced rG4 inhibits viral replication and reduces infectivity. We show that the rG4 ligands act by disrupting the unique pattern of OC43 "discontinuous transcription." Thus, rG4-targeting compounds present a potential therapeutic approach for targeting ssRNA viruses.