Page 5 - T. Protein modification and regulation

Asymmetric dimethylation of Arginine-617 of Caprin1 regulates stress granule
associated transcriptome

Sojeong Eom, Dahee Choi, Seung-hoi Koo

Division of Life Sciences, Korea University, Seoul, Korea
BACKGROUND AIM
Stress granule(SG) is a form of dynamic RNP granules, which consists of various Recent articles suggested that dynamic RNA redistribution could occur due to
mRNAs, translation factors, RNA-binding proteins like Caprin1, and non-RNA- diverse stresses. So we thought that post-translational modification of
binding proteins. In mammalian cells, When SG is induced by arsenite, Caprin1 Caprin1 RNA binding site may have impact on RNA granule transcriptome.
forms a complex with G3BP1 and colocalizes in the cytoplasmic RNP granule. Here, we found that when Caprin1 R617 mutated, asymmetric dimethylation
These formation and dynamics of SGs may have impact on mRNA localization, of Caprin1 was decreased significantly. Association of Caprin1 with G3BP1 or
translation, degradation and signaling pathway. PRMT1 was also decreased. In this study, we will delineate the impact of
caprin1 mutation on SG-associated transcriptome.
METHODS
• Preparation of RNA Samples for SG-RNA analyzation
After transfection of HA-Caprin1 WT and HA-Caprin1 R617K for 48h, cells were stressed with 500μM arsenite for 1h. After washing with media pre-warmed to 37⁰C,
cells were harvested and lysed with PLB buffer (100 mM KCl, 5 mM MgCl₂, 10 mM HEPES (pH 7.0), 0.5 % NP40, 1 mM DTT, 100 U/ml RNase Out, 1× complete
proteinase inhibitor cocktail). All subsequent steps were conducted on ice or at 4 ⁰C. resuspended cells were lysed by passing through 24G 3/4 needle, then
centrifuged at 1,000×g for 5min. After Centrifugation, 50µl of the lysate was transferred to new microcentrifuge tube as input RNA sample. Rest of lysates were
centrifuged at 17,000g×g for 20min to enrich SG cores. After resuspending pellet in 1ml PLB buffer, lysates were centrifuged at 17,000g×g for 20min again. Pellet
was resuspended in 300μl PLB buffer and spun at 850×g for 2min. The supernatant represents the SG core enriched fraction. With this fraction, we proceeded
immunoprecipitation with HA bead for 3h. beads were washed three times with washing buffer 1(20 mM Tris HCl pH 8.0, 200 mM NaCl in Nuclease free DW), once
with washing buffer 2(20 mM Tris HCl pH 8.0, 500 mM NaCl in nuclease free DW), and once with washing buffer 3(PLB buffer with 2M Urea). The beads were
resuspended with 1x proteinase K buffer(100 µg/mL Proteinase K, 2M Urea, 1X TE buffer) and incubated at 37⁰C for 15min. Then, input RNA and immunoprecipitated
RNA were purified using Trizol, chloroform, and RNeasy Plus Mini Kit(Qiazen).
RESULTS
Table 1 Figure 2 Table 2
Untreated Arsenite
Site of Methylati -6/+6 peptide (lower case r is t
gene_name Protein Name refseq on he methylated residue) A
Caprin1 GPIP137 NP_001104759 614 VSRGGSrGARGLM del_Cyt-AS %RG del_RG-AS Cluster(AS)
Caprin1 GPIP137 NP_001104759 617 GGSRGArGLMNGY Actb 0.614929108 1.16743403 -1.18907636 6
Caprin1 GPIP137 NP_001104759 §638 GGYDGYrPSFSNT Akt1 0.468199826 0.77198006 1.422266498 1
Atf4 -0.030093223 0.763751449 1.588083565 1
Myc 0.438847202 1.16457689 1.633888074 1
Table1. Caprin1 ADMA sites identified in mouse brain and mouse Src 0.243015039 0.95350823 1.410043159 1
Caprin1 Caprin1
G3BP1 G3BP1
embryo (Ailan guo et al.) DAPI DAPI
Figure 1 A Input B Hepa1c1c7 Table 2. List of Transcripts Identified in Control
f/f LKO HA-mCaprin1 WT WT R617K R614K R638K and arsenite Treated Cells. (Sim Namkoong et
As〔 Ⅲ〕
HSP90 IP : IgG IP : Caprin1 - + + + + C al) Beta-actin is used as negative control, and
HA
PRMT1 f/f LKO f/f LKO Akt1, Atf4, Myc and Src seemed to enriched in
G3BP1 *
Caprin1 IP : HA 110 WT SGs.
Asym-diMe PRMT1 100 90 R617K Figure 3
(Caprin1 size) D6A8 80 70
B Input IP : IgG IP : ADMA antibody Cells with SGs≥5 (%) 60 50 A
f/f LKO f/f LKO f/f LKO HA 40 30 25
Caprin1 G3BP1 20 **
lysates PRMT1 10 0 20 WT
R617K
WT
Caprin1 R617K 15
Caprin1 size D6A8
Asym-diMe Fold Enrichment 10 **
Figure 2. Arginie-617 mutated Caprin1 is less affected by 5 N.S.
**
PRMT1 0
Akt1 ATF4 myc Src
Figure 1. PRMT1 methylates Caprin1 in primary hepatocytes (A) Hepa1c1c7 cells were infected with Ad-GFP-Caprin1 for
(A) Immunoprecipitation of PRMT1 flox and PRMT1 liver knock- 48h, and stressed with arsenite (500μM, 30min). Figure 3. Stress granules with ariginine-617
out primary hepatocytes with Caprin1 antibody. (B) SG marker G3BP1 was stained in red, and nuclei was stained mutated Caprin1 have different RNA
Immunoprecipitation of PRMT1 flox and PRMT1 liver knock-out with DAPI. SG responses were monitored by ICC. Scale transcriptome.
primary hepatocytes with ADMA antibody. bar=50μm (B) Immunoprecipitation of WT, R617K, R614K or
R638K Caprin1 transfected Hapa1c1c7 cells with HA (A) Hepa1c1c7 cells were transfected with HA-
mCaprin1 WT and HA-mCaprin1 R617K each
antibodies. (C) Hepa1c1c7 cells were transfected with
then, stressed with arsenite (500μM, 1h). After
pcDNA3-HA-mCaprin1 WT, pcDNA3-HA-mCaprin1 R617K
CONCLUSION each and arsenite was treated (500μM, 30min). These cells immunoprecipitation with HA antibody, RNA
were fixed, and proceeded ICC with antibodies against HA in isolation and purification were proceeded.
- Caprin1 is methylated by PRMT1 Relative mRNA levels were determined by RT-
green, G3BP1 in red and DAPI for nuclei. Quantification of cells
- Methylation of Caprin1 was significantly decreased with more than five SGs after arsenite treatment was described qPCR and were normalized to input levels of
when arginine-617 mutated mRNA. *P<0.05, **P<0.01, ***P<0.001.
in bar graph. The results shown are representative of two
- Reduction of Caprin1 methylation through arginine-
617 Mutation affects SG assembly and SG- independent experiments in which more than 100cells were
associated transcriptome counted in each. *P<0.05, **P<0.01, ***P<0.001
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