Unequal distribution of N6-methyladenosine in influenza virus mRNAs.

Abstract

Influenza virus mRNA is posttranscriptionally methylated at internal adenosine residues to form N6-methyladenosine (m6A). It has been previously shown that there is an average of three m6A residues per influenza virus mRNA (R. M. Krug, M. A. Morgan, and A. J. Shatkin, J. Virol. 20:45-53, 1976). To determine the distribution of m6A in the different influenza virus mRNAs, we purffied six of the mRNAs by hybrid selection, digested them with nuclease, and determined their methylation patterns by high-pressure liquid chromatography. The amount of m6A in the different mRNAs varied from one in matrix to eight in hemagglutinin.Influenza virus has a negative-strand RNA genome composed of eight gene segments (11). With the exception of retroviruses, influenza virus is the only RNA virus which replicates in the nuclei of infected cells. Nuclear replication allows influenza virus to utilize cell functions involved in the processing of nuclear RNA precursors. In this regard, two influenza virus mRNAs, M and NS, coding for the matrix protein and nonstructural protein, respectively, are themselves spliced to form smaller mRNAs, coding for two other proteins, M2 and NS2, respectively (11). In addition to splicing, influenza virus mRNAs have been shown to contain internal N6-methyladenosine (m6A) residues. m6A is a common posttranscriptional modification found in cellular and viral mRNAs synthesized in the nucleus (5, 12) at the consensus sequences Gm6AC or Am6AC (14,18). Evidence derived from localization (6, 7) and inhibitor (3) studies suggests that this modification may play a role in mRNA metabolism.It has previously been shown that influenza virus mRNA contains an average of three m6A residues per molecule, one of which may be derived from the 5' end of the host cell mRNA used to prime viral transcription (10). To study the possible significance of m6A in influenza virus mRNA metabolism, we determined the distribution of m6A residues in six individual hybrid-selected mRNAs.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

“…Therefore, methylation at this site is nonstpichiometric. Furthermore, the methyl addition found.in both the 13-and 39-base oligonucleotides is nonstoichiom.etric and corresponds to that reported for bPRL mRNA (22) and viral RNAs (18,21). One of the alternatives which may give rise to this nonstoichiometry is that the methyltransferase is influenced by the sequence context in which the consensus motif is located.…”

“…Furthermore, as this region of RNA will be lost during processing, m6A at specific locations in precursor RNA is not conserved during maturation. Previous investigators exam- (9), and influenza virus mRNAs (21). The detailed kinetic studies of m6A in adenovirus nuclear and cytoplasmic RNAs demonstrated m6A conservation during mRNA formation (9,33).…”

“…1A) had been bound (21). Following hybridization, filters were treated with RNase T1 to remove unhybridized RNA and washed and the RNA was eluted (22 (Du Pont Co.), and analyzed by fluorography as described previously (22).…”

Viral Epitranscriptomics

“…However, we now know that many cellular mRNAs, including mRNAs encoding housekeeping genes, lack any m 6 A residues, while highly regulated mRNAs may contain 10 or more (2,3). The first demonstration of m 6 A residues on viral mRNAs soon followed and, using the biochemical approaches available at that time, a range of mRNAs encoded by several nuclear DNA and RNA viruses were then shown to bear fairly high levels of m 6 A, with the eight influenza A virus (IAV) mRNAs bearing an average of three m 6 A residues each (4)(5)(6)(7)(8)(9). Subsequent work looking at each individual IAV mRNA revealed that IAV mRNAs actually contain from 1 to 8 m 6 A residues each (Table 1) (5).…”

“…Of these, by far the most prevalent internal modified base found on mRNAs is m 6 A, and recent work has now begun to reveal how m 6 A affects mRNA function and how to precisely map the m 6 A residues present on mRNAs (1)(2)(3). m 6 A is also highly prevalent on a wide range of different viral RNA species (4)(5)(6)(7)(8)(9)(10)(11)(12)(13), and recently, the first reports demonstrating a significant phenotypic effect of these m 6 A modifications have been published (10)(11)(12)(13)(14). Therefore, we will focus this review entirely on m 6 A and how this particular modification might affect different aspects of the viral life cycle.…”