Identification and Characterization of a Novel Group of Natural Anti-Sense Transcripts from RNA1.2 Gene Locus of Human Cytomegalovirus
Natural anti-sense transcripts (NATs) are RNA molecules transcribed from the complementary DNA strand of annotated genes. These transcripts play significant roles in regulating gene expression at various stages, including pre-transcriptional, transcriptional, and post-transcriptional levels. Despite their widespread presence across different species, the anti-sense transcription of the RNA1.2 gene locus in human cytomegalovirus (HCMV) had not been investigated prior to this study. This research aimed to identify and characterize NATs from the RNA1.2 gene locus in a clinically isolated HCMV strain.
Human cytomegalovirus is a member of the beta-herpesvirus family, with a genome spanning 230 to 240 kilobases. HCMV infection is generally asymptomatic in healthy individuals but can cause severe complications in immunocompromised patients and neonates. The virus encodes a variety of RNAs, including non-coding RNAs (ncRNAs), which have been increasingly recognized for their critical roles in viral life cycles and immune responses. Among these, RNA1.2 is one of the most abundant viral transcripts, accounting for a significant portion of polyadenylated viral RNA. Although RNA1.2 is primarily considered a long non-coding RNA (lncRNA), recent evidence suggests it may also encode a polypeptide.
The study utilized strand-specific high-throughput RNA sequencing (RNA-seq) to identify potential anti-sense transcripts (ASTs) from the RNA1.2 gene locus. RNA was extracted from human embryonic lung fibroblasts (HELFs) infected with the HCMV clinical isolate HAN. The RNA-seq data were analyzed and visualized using the Integrative Genomics Viewer (IGV) software. To confirm the presence of these transcripts, Northern blotting and rapid amplification of cDNA ends (RACE) were employed.
The RNA-seq analysis revealed transcription from the opposite strand of the RNA1.2 gene locus, indicating the presence of NATs. Three distinct HCMV NATs, named RNA1.2 AST1, RNA1.2 AST2, and RNA1.2 AST3, were characterized. These transcripts were found to be oriented from the complementary strand of the RNA1.2 locus and were predominantly expressed during the late phase of HCMV infection. The 5′ and 3′ termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene.
Northern blotting confirmed the presence of these NATs, with three bands of approximately 1100, 1000, and 600 nucleotides detected in the 72-hour post-infection (hpi) RNA sample. No such bands were observed in the 24 hpi, 48 hpi, or uninfected RNA samples, suggesting that these NATs are specifically induced during the late phase of HCMV infection. Additionally, a transcript of approximately 1.2 kilobases was detected using a probe specific to RNA1.2, confirming the transcription of the sense strand.
RACE analyses were conducted to determine the precise 5′ and 3′ ends of the RNA1.2 ASTs. The 3′-RACE results showed two predominant bands of about 400 and 250 base pairs, with sequencing revealing that the 3′ ends of the RNA1.2 ASTs were located at nucleotide positions 8062 to 8093 and 7843 to 7855, downstream of a consensus polyadenylation signal (AATAAA) at positions 7843 to 7848. The 5′-RACE analysis identified three predominant bands of about 500, 350, and 250 base pairs, with sequencing indicating that the RNA1.2 ASTs initiated at nucleotide positions 7479, 7331, and 7203 of the HCMV HAN genome. These initiation sites are downstream of a putative “TATA” box starting at nucleotide position 7074.
The study identified at least three distinct RNA1.2 ASTs, each with different 5′ and 3′ termini. These transcripts are internal NATs, as they are fully covered by the sense transcript of the RNA1.2 gene. The RNA1.2 ASTs are polyadenylated, as confirmed by the 3′-RACE results, and are localized in the cytoplasm, where they may interact with their overlapping sense RNAs to regulate gene expression. The abundance of RNA1.2 ASTs was found to be much lower than that of RNA1.2, consistent with the general observation that NATs are typically less abundant than their sense counterparts.
The differential transcription termination sites of the RNA1.2 ASTs suggest complex regulatory mechanisms. While RNA1.2 AST3 terminates at a canonical polyadenylation signal, RNA1.2 AST1 and RNA1.2 AST2 terminate over 250 nucleotides downstream of the same signal, potentially indicating read-through transcription. This phenomenon has been observed in other herpesviruses and may contribute to the diversity of viral transcripts.
The initiation sites of the RNA1.2 ASTs are located downstream of a putative “TATA” box, a common promoter element in eukaryotic genes. The presence of high CG content upstream of the transcription start site (TSS) of RNA1.2 AST1 suggests that different regulatory mechanisms may be involved in the initiation of these transcripts. The study hypothesizes that RNA1.2 ASTs may play a role in regulating the expression of the RNA1.2 gene, although further investigation is required to elucidate their precise functions.
In conclusion, this study identified and characterized a novel group of NATs from the RNA1.2 gene locus of HCMV. These transcripts are differentially regulated and may play a role in the viral life cycle by modulating the expression of the RNA1.2 gene. The findings provide a foundation for future research into the biological functions of these NATs and their potential implications for HCMV infection.
doi.org/10.1097/CM9.0000000000000299
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