RNA最新文献

In addition to their function in protein synthesis, translating ribosomes serve as sensors that communicate the presence of aberrant messenger RNAs (mRNAs); however, how they recognize damage to their ribosomal RNA (rRNA) remains poorly understood. The conserved sarcin/ricin loop (SRL) of the 25S rRNA is a component of the GTPase center essential for ribosome movement during translation. In this study, we expressed an RNA N-glycosylase called pokeweed antiviral protein (PAP) in yeast Saccharomyces cerevisiae to specifically damage rRNA by hydrolysis of a purine base from the SRL. 25S rRNA depurination inhibited translation elongation, as shown by reduced incorporation of a methionine analog and binding of eukaryotic elongation factor 2 (eEF2) to ribosomes. PAP expression altered sucrose gradient profiles, increasing free subunits and 80S peaks and reducing polysomes without causing ribosome collisions. We discovered depurinated rRNA associated with 80S monosomes and polysomes, suggesting that cells would detect damage to rRNA during active translation. These ribosomes were ubiquitinated by E3 ligases Mag2 and Hel2, elements of the 18S nonfunctional rRNA decay (NRD) pathway involved in recognizing slow-moving ribosomes. Furthermore, mass spectrometry analysis revealed ubiquitination of ribosomal protein uS3, characteristic of 18S NRD. Even though the SRL is a component of the large ribosomal subunit, its depurination is signaled by ubiquitin ligases that recognize damage to the small subunit. We suggest that slow translation elongation is the factor that communicates SRL depurination to E3 ubiquitin ligases, which extends our understanding of how rRNA integrity is surveilled in yeast.

Human papilloma viruses (HPV) are linked to cancers, but how virus-carrying tumor cells express HPV-encoded antigens without attracting the immune system is still poorly understood. Here, we show how low- and high-risk HPV types equally exploit a cis-acting mechanism to limit the translation of the E6 mRNA, reducing the production of antigenic peptide substrates for the major histocompatibility class I (MHC-I) pathway. Introducing particular combinations of preferable codons throughout the HPV-16 E6 mRNA promotes mRNA translation and production of antigenic peptide substrates in mammalian cells but has minimal impact on E6 synthesis in Saccharomyces cerevisiae Using a gradual synonymous codon exchange, we identified a codon series with a significant effect on E6 translation rate. Unexpectedly, changing four nonpreferable codons to preferable codons in the wild-type sequence resulted in an ∼50% reduction in E6 expression. However, five additional changes to preferable codons further upstream shifted this inhibition to a strong induction of E6 expression, while they had no effect when introduced alone. These findings suggest a nuanced relationship between tRNA pools and translation rate, emphasizing how HPV uses codon usage to evade immune detection.

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.

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.

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.

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.

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.