RESUMO
The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.
Assuntos
RNA , Sequências Reguladoras de Ácido Nucleico , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Proteínas/genética , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade de RNA , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Telomeres are part of a highly refined system for maintaining the stability of linear chromosomes. Most telomeres rely on simple repetitive sequences and telomerase enzymes to protect chromosomal ends; however, in some species or telomerase-defective situations, an alternative lengthening of telomeres (ALT) mechanism is used. ALT mainly utilises recombination-based replication mechanisms and the constituents of ALT-based telomeres vary depending on models. Here we show that mouse telomeres can exploit non-telomeric, unique sequences in addition to telomeric repeats. We establish that a specific subtelomeric element, the mouse template for ALT (mTALT), is used for repairing telomeric DNA damage as well as for composing portions of telomeres in ALT-dependent mouse embryonic stem cells. Epigenomic and proteomic analyses before and after ALT activation reveal a high level of non-coding mTALT transcripts despite the heterochromatic nature of mTALT-based telomeres. After ALT activation, the increased HMGN1, a non-histone chromosomal protein, contributes to the maintenance of telomere stability by regulating telomeric transcription. These findings provide a molecular basis to study the evolution of new structures in telomeres.
Assuntos
Células-Tronco Embrionárias Murinas/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Telomerase/genética , Homeostase do Telômero/genética , Telômero/genética , Animais , Proteínas de Ligação a DNA/genética , Epigenômica/métodos , Células HEK293 , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Telomerase/metabolismo , Telômero/enzimologia , Fatores de Transcrição/genéticaRESUMO
TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.