Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres (CENs) via histone H3 variant CENP-A and low levels of CEN transcripts. RNA polymerase II (RNAPII) activity is restrained by the CEN histone code, while CEN RNA concentrations are reduced by the nuclear exosome. Using S. cerevisiae, we add kinase Rio1 to this scheme as it downregulates RNAPII, and promotes CEN RNA turnover via exoribonuclease Rat1. Transcription factor Cbf1 and the assembled kinetochore further restrain CEN transcription. CEN transcripts exist as long (up to 11,000nt) and short RNAs (119±40nt), which may underlie CEN identity and kinetochore recruitment. While also curtailed by Rio1, Rat1, and the exosome, periCEN RNAs (<200nt) accumulate at levels that are one order of magnitude higher than the CEN transcripts. Depleting Rio1 causes CEN and periCEN RNA buildup, kinetochore malformation, and chromosome loss. Depleting human orthologue RioK1 leads to CEN RNA accumulation and micronuclei formation, suggesting that Rio1/RioK1 activity at centromeres is conserved.

APOBEC-mediated Editing of SARS-CoV-2 Genomic RNA Impacts Viral Replication and Fitness

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more virulent and evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Recent SARS-CoV-2 sequence analyses showed a strong bias toward C-to-U mutation, suggesting that host APOBEC cytosine deaminases with immune functions may cause the mutation. We report the experimental evidence demonstrating that APOBEC3A and APOBEC1 can efficiently edit SARS-CoV-2 RNA to produce C-to-U mutation at specific sites. However, APOBEC-editing does not inhibit the viral RNA accumulation in cells. Instead, APOBEC3A-editing of SARS-CoV-2 promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the unpredictability of random mutations, this study has significant implications in predicting the potential mutations based on the UC/AC motifs and surrounding RNA structures, thus offering a basis for guiding future antiviral therapies and vaccines against the escape mutants.

miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections

ABSTRACT: A liver-specific microRNA, miR-122, anneals to the HCV genomic 5’ terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full length RNAs with specific mutations in the 5’ UTR can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having EMCV IRES-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication when compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by amount of viral RNA delivered. HCV RNAs replicating independent of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of a miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection, and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE: The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as potential HCV therapy.

Heterogeneity in the Response of Different Subtypes of Drosophila melanogaster Midgut Cells to Viral Infections

Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Here, public scRNA-seq datasets from Drosophila melanogaster midgut cells were used to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication.

miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections

A liver-specific microRNA, miR-122, anneals to the HCV genomic 5’ terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full length RNAs with specific mutations in the 5’ UTR can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having EMCV IRES-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication when compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by amount of viral RNA delivered. HCV RNAs replicating independent of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of a miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection, and also supports new replication complex formation during ongoing infection and after infected cell division.

PARP dependent acetylation of N4-cytidine in RNA appeared in UV-damaged chromatin

Posttranscriptional RNA modifications, including the presence of methyl-6-adenosine (m6A), methyl-5-cytosine (m5C), or pseudo-uridine (Ψ), are known for over many years, but their functional properties have not been fully elucidated yet. Similarly, the regulatory role of N4-cytidine (ac4C) acetylation in RNA must be explored. Here, we observed PARP-dependent accumulation of ac4C RNA at UVA-microirradiated chromatin, which appears 2-5 minutes after genome injury, simultaneously with m6A RNAs but with distinct kinetics. When m6A RNAs disappeared from the lesions, the high level of ac4C RNA was maintained up to 20 minutes after genome injury. Surprisingly, the process of ac4C RNA accumulation at DNA lesions was not accompanied by the recruitment of acetyltransferase NAT10 to UVA-induced DNA lesions. This process was PARP dependent, and data show how epitranscriptomic features can contribute to DNA damage repair.

Molecular Determinants for RNA Release into Extracellular Vesicles

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

N6 -Methyladenosine Negatively Regulates Human Respiratory Syncytial Virus Replication

N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and several viral RNA including human respiratory syncytial virus (HRSV). Here, we evaluated the impact of m6A writers, erasers and readers on HRSV genomic RNA accumulation and inclusion bodies assembly during viral replication. We observed that the METTL3/METTL14 m6A writer complex plays a negative role in HRSV protein synthesis and viral titers, while m6A erasers FTO and ALKBH5 had the opposite effect. We also observed that m6A readers YTHDF1-3 bind to the viral genomic RNA inducing a decrease in its intracellular levels and thus, inhibiting viral replication. Finally, we observed that overexpression of YTHDFs proteins caused a decrease in the size of inclusion bodies (IBs), accompanied by an increase in their number. METTL3 knockdown cells showed an opposite effect indicating that the dynamics of IBs assembly and coalescence are strongly affected by m6A readers in a mechanism dependent on m6A writers. Taken together, our results demonstrated that the m6A modification negatively affects HRSV replication, possibly through a mechanism involving the assembly of inclusion bodies, the main factories of viral genomic RNA synthesis.

Phase separation of a plant virus movement protein and cellular factors support virus-host interactions

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.

PARP dependent recruitment of RNA methylated at 8-adenosine is linked to base excision repair mechanism

Methylation of RNAs, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we observed that 8-methyladenosine (m8A)-modified RNA is recruited to UVA-microirradiated chromatin, which was reduced by inhibiting both DNA methylation and histone acetylation, especially in later phases of DDR. Most importantly, clinically used PARP inhibitor (PARPi), olaparib, prevents both m8A and m6A RNA accumulation at microirradiated chromatin. Testing the effect of PARPi on the efficiency of BER, NHEJ, and HR repair pathways, we observed that NHEJ repair proteins are down-regulated after PARP inhibition and recruitment of XRCC1, a factor of BER, to DNA lesions was abolished entirely. Conversely, the PARP inhibitor, olaparib, enhanced the genome-wide level of γH2AX that significantly interacted with m8A RNA, similar to DNA. Together, we showed that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent, similarly as XRCC1 playing a role in the BER mechanism. We found that γH2AX likely stabilizes m8A/m6A RNA-DNA hybrid loops that are formed during PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.