We previously described methods for using lacZ
-based vectors to screen for mutations in regulated genes (25
). The present study characterized one of these mutant cell lines, termed 7.4.2, generated by an inserted U3βGeo provirus. The 7.4.2 cell line showed an increase in lacZ
expression upon differentiation of ES cells into embryoid bodies (28
). We show here that the gene disrupted by the provirus encodes the murine arginine methyltransferase 1 enzyme (EC 18.104.22.168
). Embryos homozygous for the mutation arrest in development prior to E6.5, indicating that protein methylation is required for early postimplantation development. However, cell lines derived from homozygous mutant embryos are viable despite the fact that Prmt1 enzyme levels and the extent of protein methylation are significantly reduced.
cDNAs encoding Prmt1 were characterized as part of our analysis of the 7.4.2
mutation. As previously reported for the human gene, Prmt1
transcripts are alternatively spliced to generate coding sequences for proteins of 353 or 371 amino acids. Overall, murine Prmt1 shares 100 and 95% sequence identity with the rat and human proteins, respectively. Phylogenetic conservation of the two proteins suggests that each has a distinct function, possibly related to substrate specificity or regulation of enzyme activity. Finally, Prmt1
was mapped to mouse chromosome 7 in a region syntenic with the region of human chromosome 19q, where the human gene had been previously mapped (29
). Neither region is associated with disease loci in mice or humans.
Our analysis of Prmt1
expression confirms earlier reports describing widespread expression of the orthologous rat and human genes in all of the tissues examined (17
). In addition, all of the tissues examined expressed both spliced forms of the murine gene. Expression of the inserted β-galactosidase marker was highest in developing neural structures. This is potentially significant in light of previous studies showing high levels of methyltransferase activity in the developing brain that decline after birth (22
). However, homozygous mutant embryos die close to the time when the Prmt1
gene is first induced, as assessed by β-galactosidase staining, and well before the onset of neural development. As early embryonic death appears to be a common consequence of mutations disrupting basic cellular processes (7
), the phenotype is consistent with the important role Prmt1 is thought to play in RNA metabolism. For example, the timing of embryonic death is similar to that observed with a mutation in Fug1
, which encodes the RAN GTPase-activating protein (8
), and is earlier than that observed with a mutation in hnRNP C, a highly abundant, ubiquitous constituent of nuclear riboprotein complexes (35
). Both RAN GAP and hnRNP C appear to function in RNA biogenesis and transport.
By comparing the levels of enzyme activity in extracts from wild-type and Prmt1
-deficient cells, we showed that Prmt1 accounts for over 85% of the total arginine methyltransferase activity in ES cells, as assayed using an optimal (21
) synthetic RGG substrate. However, Prmt1 is responsible for just over 50% of the normal steady-state levels of NG
-dimethylarginine present in cellular proteins. Therefore, other arginine methyltransferases, presumably with different substrate specificities, appear to make significant contributions to the total dimethylarginine content of the cell. Such enzymes could include PRMT3 and HRMT1L1, type I-related enzymes that modify RGG substrates poorly, if at all (29
The methylation status of cellular proteins was also assessed by testing their capacity to be methylated in vitro. Prmt1, present in wild-type cell extracts, was incubated with proteins isolated from either wild-type or Prmt1-deficient cells in the presence of S
H]methionine. While proteins from wild-type cells had negligible acceptor activity, proteins from Prmt1-deficient cells were significantly hypomethylated. This has two implications. First, the function of Prmt1 appears to be nonredundant, since other cellular enzymes compensate for no more than a small part of the lost Prmt1 activity. Second, as previously observed with nucleolin and fibrillarin (18
), most potential substrates in normal cells appear to be blocked by prior methylation. Our analysis does not exclude the possibility that individual substrates are present in a hypomethylated state in ES cells or that the methylation status may change in response to different physiological conditions. Hypomethylated proteins may also exist only transiently, for example, during a specific step in a biochemical process. However, this latter possibility would require an arginine demethylase, an activity that has not yet been identified in mammalian cells. Experiments to study potential dynamic changes in protein methylation are in progress.
In summary, this report describes the first mutation in a mammalian arginine methyltransferase. These enzymes have been implicated in a wide variety of cellular processes, including cell proliferation, signal transduction, and protein trafficking. The availability of Prmt1-deficient cells will assist efforts to identify physiological substrates and to understand the function of the enzyme in normal cellular metabolism.