METTL14 and FTO mediated m⁶A modification regulate PCV2 replication by affecting miR-30a-5p maturity

Figures & data

Table 1. RT-Qpcr Primer sequence.

Primer Name	Sequence (5’-3’)
Sus-METTL14-F	AGAAACTTGCAGGGCTTCCT
Sus-METTL14-R	TCTTCTTCAGGCAAATTTTCTT
Sus-METTL3-F	CTGGGGTTACGAACGGGTAG
Sus-METTL3-R	TGACACCAACCAAGCAGTG
Sus-WTAP-F	GGCGAAGTGTCGAATGCT
Sus-WTAP-R	CCAACTGCTGGCGTGTCT
Sus-FTO-F	GACGGGAGCCTTGGATTACA
Sus-FTO-R	AAACTCGACCTCGTTGTGGAT
Sus-ALKBH5-F	CCCGAGGGCTTCGTCAACA
Sus-ALKBH5-R	CGACACCCGAATAGGCTTGA
Sus-YTHDF2-F	GGCAGCACTGAAGTTGGG
Sus-YTHDF2-R	CTATTGGAAGCCACGATGTTA
β-actin-F	TCTGGCACCACACCTTCT
β-actin -R	ATCTGGGTCATCTTCTCAC

Table 2. siRNA target sequence.

Primer Name	Sequence (5’-3’)
FTO-sus-143	GGATCCAAGGCAAAGATTTGC
FTO-sus-1260	GCTGTGCTTCGTGAAGTTAGA
FTO-sus-735	GCGTGTATAGGTCCAGATTTC
METTL14-sus-255	GCATTGGTGCTGTGTTAAATA
METTL14-sus-854	GGACTTGGGATGACATCATGA
METTL14-sus-1379	GGGAATCAAAGGAACTGTTAA

Table 3. miRNA quantitative analysis primer sequence.

Primer Names		Sequence (5’-3’)
miR-328	Sense	GCTGGCCCTCTCTGCCC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-504	Sense	GCGAGACCCTGGTCTGCACT
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-7134-5p	Sense	CGATGTCCGCGGGTTCC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-30b-3p	Sense	GCGCTGGGAGGTGGATGT
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-125a	Sense	GCGTCCCTGAGACCCTTTAA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-92b-3p	Sense	GCGTATTGCACTCGTCCCG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-10b	Sense	CGCGTACCCTGTAGAACCGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-155-5p	Sense	GCGCGTTAATGCTAATTGTGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-125a	Sense	GCGTCCCTGAGACCCTTTAA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-92b-3p	Sense	GCGTATTGCACTCGTCCCG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-10b	Sense	CGCGTACCCTGTAGAACCGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-155-5p	Sense	GCGCGTTAATGCTAATTGTGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-21	Sense	CGCGCGTAGCTTATCAGACTG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-29b	Sense	CGCGTAGCACCATTTGAAATC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-361-3p	Sense	GCCCCCAGGTGTGATTCTG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-15a	Sense	GCGCGTAGCAGCACATAATG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-30a-5p	Sense	CGCGTGTAAACATCCTCGAC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT

Table 4. Pri-miRNA quantitative analysis primer sequence.

Primer Names		Sequence (5’-3’)
Sus-pri-miR-328	Sense	ATGGAAGCTCTGGTCTTGGC
	Anti-Sense	GGCACACACTTTCTCCCTGA
Sus-pri-miR-504	Sense	ACAGCCATTTACACGGGGTT
	Anti-Sense	TCAGTCTCCGCAAAAAGGCA
Sus-pri-miR-7134-5p	Sense	AGGTGAGACTTGAGTGAGGAGA
	Anti-Sense	GCCCTTATGCAAGACAGCGG
Sus-pri-miR-30b-3p	Sense	AGTCTCAGGTAGAGGGCCAG
	Anti-Sense	ACGGTTGATATTCCAAGTCAGC
Sus-pri-miR-125a	Sense	TTGTGCCGCAGCCCC
	Anti-Sense	GGTTTTCAGGGGAGGAAGGG
Sus-pri-miR-92b-3p	Sense	CGCGGTGCAGTGTTGTTCTTT
	Anti-Sense	GCGGCGACACTCCAGTCTAT
Sus-pri-miR-10b	Sense	ACAGCCAGAATCGGAAGTGG
	Anti-Sense	TTTTGGCCTTGCATTCCTGC
Sus-pri-miR-128	Sense	ACTGTGTTGTCTGTTGAAGCC
	Anti-Sense	TCACTGCTTATATGGCTCCAGT
Sus-pri-miR-155-5p	Sense	CTCTTGCAGGTGGTGCAAAC
	Anti-Sense	TGTAGGAGTCAGACGGAGGT
Sus-pri-miR-21	Sense	TCCTTTGGGATGTTTCTGGTTGA
	Anti-Sense	TCACGACGGTAGGCAAAACA
Sus-pri-miR-29b	Sense	GCCAGCTTATTGTAGGGAAGCA
	Anti-Sense	CCTTCAAAGTCTTCAGGACCG
Sus-pri-miR-31-3p	Sense	CTGGGATTTGGGAGCTTATCA
	Anti-Sense	GTAGAATGGGTACATGGAGATCAG
Sus-pri-miR-15a	Sense	TATCTTTAGGTGGTAAGGTGTGTT
	Anti-Sense	AGGCACTGCTGACATTGCTA
Sus-pri-miR-30a-5p	Sense	ACCCTTCTCCAGGGTAACCA
	Anti-Sense	AACATCCGACTGAAAGCCCG
Sus-pri-miR-122	Sense	TATGGTGGAATGTGCAGGTGAA
	Anti-Sense	AACCTTCTTGCCCATACCAAAC
Sus-pri-miR-98	Sense	CTGTCGTCAGGTAAAATGGCTC
	Anti-Sense	TGATGGAGGCAGCGGTATCAA

Table 5. The mimic sequence of miR-30a-5p is shown in the table.

Names		Sequence (5’-3’)
miR-30a-5p mimics	Sense	UGUAAACAUCCUCGACUGGAAG
	Anti-Sense	CUUCCAGUCGAGGAUGUUUACA
NC	Sense	UCACAACCUCCUAGAAAGAGUAGA
	Anti-Sense	UCUACUCUUUCUAGGAGGUUGUGA

Figure 1. PCV2 infection affects the expression level of m⁶A methylation in PK-15 cells.

Note: (a) Heat map of intracellular m⁶A methylation-related gene analysis after PCV2 infection. (b) The expression changes of each methylase in PK-15 cells after PCV2 infection. (c) The m⁶A content of total RNA in PK-15 cells infected with PCV2 was compared with that in normal PK-15 cells (p < 0.05). (d) RT-qPCR was used to detect the expression of m⁶A regulation-related genes in PK-15 cells infected with the PCV2 virus. β-actin was used as an internal reference. (e) m⁶A regulation-related genes expression of PCV2 virus at 72 h in PCV2 virus-infected and uninfected groups and ACTB as an internal control. The ratios to ACTB from three independent experiments are shown (*, P, 0.05; **, P, 0.01; ***, P, 0.001). ImageJ was used to quantify the level of protein.

Figure 2. METTL14 and FTO regulate the level of m⁶A methylation in PK-15 cells. (a, b) METTL14 and FTO knockdown efficiency in each of three siRNA-transfected PK-15 cells at 48 h and 72 h post-transfection. β-actin served as a loading control in the western blot. Western blotting of METTL14 and FTO. (c).

Figure 3. METTL14 and FTO regulate PCV2 replication in PK-15 cells. (a) RT-qPCR detection of PCV2 mRNA expression at 48 h and 72 h in the overexpression group (b) RT-qPCR detection of PCV2 virus in the siRNA group, (c) Cap protein expression of PCV2 virus at 48 h and 72 h in oe-pc3.1-METTLE14, oe-pc3.1-FTO, si-METTLE14, si-FTO and PK-15 cells of the control group, (d) RT-qPCR detection of miRNA expression at 72 h in the PCV2 virus-infected and uninfected groups.

Figure 4. miRnas were quantified by RT-qPCR upon METTL14 and FTO depletion or overexpression in PK-15 cells after PCV2 infection.

Figure 5. Pri-miRnas were quantified by RT-qPCR upon METTL14 and FTO depletion or overexpression in PK-15 cells after PCV2 infection.

Figure 6. MiR-30a-5p promotes PCV2 replication. (a) western blot detection of cap protein expression in PK-15 cells infected with PCV2 under the action of miR-30a-5p mimics. (b) Western blot detection of cap protein expression in PK-15 cells infected with PCV2 under the action of miR-30a-5p mimics, oe-FTO, and si-METTL14.

Data Availability statement

The data that support the findings of this study are available from the corresponding author, [[email protected]], upon reasonable request.