PRMT5 is the predominant Type II arginine methyltransferase, indicating that it confers both MMA and SDMA marks on target substrates in a distributive manner.
The primary targets of PRMT5 methylation are RNA-binding proteins, epigenetic modulators and core histones, which has implicated this enzyme in transcriptional regulation and the control of faithful alternative splicing . PRMT5 is not enzymatically active on its own, and is found in a protein complex called the methylosome.
Regardless of the methylation target, PRMT5 requires the co-factor methylosome protein 50 (MEP50) for stability and enzymatic activity . MEP50 is also referred to as WDR77. Loss of MEP50 results in the destabilization of the PRMT5 protein and vice versa . Enzymatic activity is further dependent on hetero-octamerization of these two proteins to form a complex of four PRMT5 molecules and four MEP50 molecules . MEP50 has also been identified as a potential coactivator of the androgen receptor , but it is unclear whether PRMT5 is recruited with MEP50 in this context, or whether it functions independently. Importantly, HeLa cell fractionation studies from a sucrose gradient indicate that PRMT5 and MEP50 only occur together and are not found in a complex without the other, nor do they exist in the free un-complexed form . Similar fractionation experiments using Xenopus egg extracts and gel filtration also reveal the existence of a single PRMT5–MEP50 complex and no free monomeric form of either protein . Thus, these two proteins are tightly complexed and likely do not function independently.
The PRMT5–MEP50 protein complex requires additional adaptor proteins to aid in identifying substrates that will be targeted for symmetric methylation. There are five known adaptors that link the methylosome to its substrates, and these are pICln, RIOK1, COPR5, Sharpin and OXR1A. pICln is a spliceosome assembly chaperone, which recruits PRMT5 to facilitate the efficient methylation of SmB/B’ and SmD1/2 , as well as a number of ribosomal proteins . A second adaptor is RIOK1, which is critical for the methylation of nucleolin by PRMT5–MEP50 , and is important for pre-rRNA transcription and processing. RIOK1 and pICln compete for binding, suggesting that there may be a common pocket for adaptor protein binding, either on PRMT5 or MEP50 . COPR5 is the third adaptor to be identified, and it recruits PRMT5 activity to nucleosomes to support the deposition of H3R8me2s and H4R3me2s marks, in its role as an epigenetic regulator . The fourth adaptor is Sharpin, and this interaction targets PRMT5 to methylate the transcription factor SKI . Finally, OXR1A also regulates PRMT5′s ability to methylate histones, and it is the H3R2me2s methylation that is stimulated by this adaptor . OXR1A and PRMT5 interact in the pituitary gland and regulate growth hormone expression, which in turn impacts liver metabolism. Both RIOK1 and pICln were identified in independent shRNA screens that also identified PRMT5 as a vulnerability in MTAP-null tumors , further supporting the key role that these adaptors play in helping the PRMT5–MEP50 methylosome find its targets for methylation.
The initial characterization of PRMT5 as an arginine methyltransferase revealed that it methylates H2A and H4, using an in vitro methylation assay . Importantly, the first five residues of H2A and H4 are identical (SGRGK…), and it is the arginine in position 3 that is methylated by PRMT5. Knockout studies showed that the H2AR3me2s modification is particularly sensitive to PRMT5 loss in vivo . Sm proteins were also shown to be methylated by PRMT5 early on in the study of this PRMT . Since then, over the last twenty years, a large number of PRMT5 substrates have been identified . These studies have been spurred on by the development of efficient pan-substrate antibodies that recognize Rme2s marks on different substrates, and can be used to enrich for methylated peptides from tissue and cell extracts, which can then be identified by mass spectrometry. The first such substrate screens were performed by the Richard lab , and subsequent screening studies have dramatically expanded on the number of known symmetrically methylated proteins into the 100s . Gene ontology (GO) analysis of the identified PRMT5 substrates reveals strong enrichment of RNA splicing and processing, as well as PTM regulated gene expression pathways and, to a lesser extent, translation.
The PRMT5 field has been confounded by the occurrence of a major artifact of tandem affinity protein (TAP) complex purifications that use the FLAG-tag. Over the years, it is well established that when purifying a FLAG tagged protein using FLAG-M2 beads, a major contaminant is the PRMT5–MEP50 protein complex, because the M2 antibody binds directly to PRMT5 and purifies both it and its associated proteins. This was first reported by Danny Reinberg’s lab over 18 years ago . Subsequent studies by the Siekhattar lab reported the same thing . PRMT5 is also listed as a common contaminant of FLAG immunoprecipitation experiments . Most recently, the CRAPome was published, which highlights the major problems with affinity purification-mass spectrometry data sets . Indeed, they showed that 94% of all FLAG purifications data sets detect PRMT5 peptides. Thus, the misassignment of PRMT5 in many FLAG-tagged protein complex purifications has led many researchers astray, and these artifacts have found their way deep into the published literature.
It is very likely that loss of PRMT5 and MEP50 in mice will phenocopy each other, as they are codependent on each other for protein stability. Indeed, the interdependence and essentiality of MEP50 and PRMT5 complexing is supported by the fact that the mouse knockouts of both PRMT5 and MEP50 result in early lethal developmental defects. MEP50 knockout mice display an early embryonic lethal phenotype with no null embryos detected at E8.5 . The PRMT5 knockout mice also display a very early embryonic lethal phenotype .
The early lethality of these total knockouts has made it necessary to generate conditional alleles for both PRMT5 and MEP50, to help elucidate the biological roles of this protein complex in vivo. Importantly, conditional knockouts of PRMT5 have provided additional insights into its roles in T and B cell development, limb development and neural development . A conditional allele for the study of MEP50 loss in adult mice is also available, but has only been used in two studies related to prostate development  and lung development , although the conditional knockout was performed ex vivo in the latter study.
The first conditional PRMT5 knockout mice were generated by crossing PRMT5fl/fl and Nestincre mice, which resulted in postnatal lethality in all homozygous null mice, and implicated PRMT5 in neuronal development . Further exploration determined that this mortality was linked to splicing variations of Mdm4, which induces a p53 response, leading to severe cranial abnormalities. Subsequently, PRMT5 was conditionally knocked out in oligodendrocytes, using Olig1cre, and identified as a key factor for myelination . Myelin basic protein has long been known to be a robust substrate for PRMT5 in in vitro studies, and the myelination defect in the conditional knockout mice provides in vivo evidence for the functional importance of this PTM .
A number of additional conditional knockouts of PRMT5 have been performed in adult mice. PRMT5 is also essential for the initiation and maintenance of hematopoiesis . Methyl-transferase localization appears to impact the modification of splicing machinery, whereas loss of PRMT5 results in alternative splicing defects via intron retention and exon skipping, which is critical for hematopoietic stem cell quiescence and viability . Both conditional knockout and small-molecule inhibitor studies reveal that loss of PRMT5 has anti-tumor activity against MLL-rearranged acute myeloid leukemia (AML) likely due to hypomethylation of essential splicing factor like SRSF1 , and further vulnerability of cancer to PRMT5 loss is bestowed on the tumors that harbor driver mutation in splicing factors . Using a CD4cre, it was recently shown that PRMT5 is dispensable for late T cell development, and is required for peripheral T cell expansion and survival . The removal of PRMT5 activity from pancreatic beta cells, using the Paxcre, reveals its role on regulation of insulin expression in vivo . PRMT5 has also been shown to play a role in muscle stem cell expansion in adult mice (using Pax7cre), but does not seem important for the proliferation and differentiation of myogenic progenitor cells during embryonic development .
In a mouse embryo developmental biology setting, PRMT5 has been identified as a key for certain differentiated chondrocytes, and in this case Prxcre was used to remove PRMT5 from developing limb buds . Conditional deletion of PRMT5 in hind limbs of mice led to severe phenotypes of atrophied long bone and knee. While essential for some chondrocyte lineages, PRMT5 is dispensable for general chondrocyte maintenance in adult mice. Inactivation of PRMT5 in germ cells (using Tnapcre) results in defects in spermatogenesis , and loss of PRMT5 in the developing lung epithelial cells (using Shhcre) causes defects in branching morphogenesis .
Although PRMT5 biology has been studied extensively through conditional knockouts in both adult mice and embryos, far fewer mouse genetic studies have been performed with the other key component of the methylosome—namely MEP50. Importantly, a conditional allele for mouse MEP50 has been generated . However, it has only been used in one study, and that was to investigate the role of MEP50 in the prostate (which we mentioned earlier). Using the Probasincre mouse, MEP50 was conditionally removed from all lobes of the developing mouse prostate. This inactivation of PRMT5 had a severe inhibitory effect on prostate development during embryogenesis, which is likely mediated by the deregulation of androgen receptor (AR) target genes due to the ability of MEP50 (and likely PRMT5) to function as an AR cofactor.
While PRMT5 and MEP50 knockouts have been shown to be essential for many key developmental pathways, PRMT5 also harbors many oncogenic characteristics through its ability to repress the expression of the tumor suppressors ST7 and NM23 . Likewise, loss of E-cadherin, a characteristic of epithelial to mesenchymal transition (EMT) which is key for metastasis, is actively repressed through the binding of PRMT5 and AJUBA to SNAIL . Overexpression of PRMT5 further induces hyperproliferation of cell lines in culture . In addition, PRMT5 has been shown to be overexpressed in many different cancers including gastric , colorectal , lung , lymphoma , ovarian , melanoma , and glioblastoma . The focus of this review is on the overexpression of PRMT5 in HCC and there are numerous reports of elevated PRMT5 levels in liver cancer . Most of these published studies demonstrate that PRMT5 is overexpressed in many different cancer types, and PRMT5 overexpression correlates with aggressive tumors and poor prognosis. However, it is not clear whether PRMT5 is an oncogenic driver, or whether the elevated PRMT5 levels are an aftereffect of a transformed state. In other words, it is still unknown whether high PRMT5 expression is a cause or a consequence of cellular transformation.