The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

BACKGROUND: Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications.
RESULTS: The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes.
CONCLUSIONS: The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p in 3'UTRs, 5'UTRs and CDSs are conservative in the orthologous mammalian genes.

Background

miRNAs participate in the regulation of the expression of protein-coding genes at the post-transcriptional stage [1]. miRNAs, as a part of the RNA-induced silencing complex, bind to mRNAs and interfere with translation or promote mRNA destruction [2]. In the last two decades, properties of miRNAs and their influences on the expression of the genes involved in all key cellular processes have been established. The actions of miRNAs on the cell cycle [3], apoptosis [4], differentiation [5], and growth and development of plants [6] and animals [7] have been shown. Connections between miRNA expression and the development of various diseases have been established. miRNA concentrations change in cancer [8] and cardiovascular diseases [9]. Metabolic perturbations change miRNA concentrations in cells [10]. The aforementioned roles do not encompass all of the biological processes in which miRNAs participate, which further proves the importance of their biological functions. Despite the significant success in the study of miRNA properties, there are obstacles in identifying the target genes of miRNAs. Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs, which significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. There are more than 2,500 miRNAs in the human genome, and they are believed to act on 60% or more genes. Therefore, it is difficult to draw specific conclusions about the participation of miRNAs in specific biological processes, and until then the connections between the majority of miRNAs and their target genes will remain unknown. Recently, a set of unique miRNAs (umiRNA) were identified that have hundreds of target genes and bind to mRNAs with high affinity [11-14]. The binding sites of these umiRNAs are located in the 3’UTRs, CDSs, and 5’UTRs of mRNAs. Among these umiRNAs, miR-619-5p interacts with the largest number of target genes that have the greatest number of binding sites with complete complementarity of miR-619-5p and mRNAs. It is necessary to identify many miRNA binding sites in the mRNAs of these genes for the control of gene expression. Furthermore, it is important to control the expression of the corresponding gene complexes that are functionally associated with miRNAs. Therefore, we have studied a unique miR-619-5p that binds to the mRNAs of several hundred human and orthologous genes.

Methods

The nucleotide sequences of mRNAs of human genes (Homo sapience – Hsa) and orthologous genes (Bos mutus - The wild yak (Bmu), Callithrix jacchus – The common marmoset (Cja), Camelus dromedarius – Arabian camel (Cdr), Camelus ferus – The wild Bactrian camel (Cfe), Chlorocebus sabaeus – The green monkey (Csa), Colobus angolensis palliatus – The Angola colobus (Can), Equus caballus - The horse (Eca), Gorilla gorilla - The western gorilla (Ggo), Macaca fascicularis – The crab-eating macaque (Mfa), Macaca mulatta – The rhesus macaque (Mmu), Macaca nemestrina - Pig-tailed macaque (Mne), Mandrillus leucophaeus – The drill (Mle), Nomascus leucogenys - The northern white-cheeked gibbon (Nle), Ovis aries – The sheep (Oar), Pan paniscus - Bonobos (Ppa), Pan troglodytes – The common chimpanzee (Ptr), Papio anubis – The olive baboon (Pan), Pongo abelii - The Sumatran orangutan (Pab), Rhinopithecus roxellana – The golden snub-nosed monkey (Rro)) were downloaded from NCBI GenBank (http://www.ncbi.nlm.nih.gov) [15] in FASTA format using Lextractor002 script [11]. Nucleotide sequences of human mature miR-619-5p (GCUGGGAUUACAGGCAUGAGCC) were downloaded from the miRBase database (http://mirbase.org) [16]. The miR-619-5p binding sites in the 5’-untranslated regions (5’UTRs), the coding domain sequences (CDSs) and the 3’-untranslated regions (3’UTRs) of several genes were predicted using the MirTarget program [12]. This program defines the features of binding: a) the localization of miRNA binding sites in the 5’UTRs, the CDSs and the 3’UTRs of the mRNAs; b) the free energy of hybridization (∆G, kJ/mole). The ratio ΔG/ΔGm (%) was determined for each site (ΔGm equals the free energy of miRNA binding with its perfect complementary nucleotide sequence).

Results

The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. The mRNAs have different miRNA binding site origins, lengths, quantities, and properties. The list of miR-619-5p target genes and the positions of binding sites are outlined in Table 1. miR-619-5p is 22 nucleotides in length and is coded by an intron of the slingshot protein phosphatase 1 (SSH1) gene, which is located on chromosome 12 [17, 18]. mRNAs of 201 genes have complete complementary binding sites for miR-619-5p (ΔG/ΔGm = 100%). Therefore, the energy of interaction of miR-619-5p with mRNA of all the genes listed in the table is the same and equal to ΔG = −121 kJ/mole.

Table 1

Positions of miR-619-5p binding sites and disease or function of target genes

Gene	Site, nt	Disease or function	PMID	Gene	Site, nt	Disease or function	PMID
ACSL6	4639	prostate cancer	19064571	MRPS25	1609	uncharacterized	26302410
ADAL	2041	proliferation	23645737	MSH3	4139	carcinogenesis	24934723
ADAM17	3466	breast cancer	22967992	NANOS1	3219	retinoblastoma	25100735
AGMAT	2207	renal carcinoma	14648699	NCMAP	2259	uncharacterized
AK1	1449	hypertension	23863634	NDUFAF7	1697	leukemia	24292274
AKT2	4571	neuroblastoma	23468863	NDUFC2	1646	colon cancer	25804238
ALDH3A2	2617	detoxification	9829906	NLN	4215	Parkinson’s D.	25378390
ANKRD16	2165	breast cancer	20453838	NRIP2	2075	atopic asthma	17075290
AP5B1	4316	differentiation	15146197	NSL1	3063	kinetochore-protein	16585270
ARGFX	2642	development	20565723	NXPE3	7447	hepatocarcinoma	26883180
ARHGEF39	1307	tumorogenesis	22327280	OPTN	2332	glaucoma	26302410
ARL11	1033	tumorogenesis	18337727	PAG1	8156	prostatic cancer	21092590
ATCAY	2991	schizophrenia	19165527	PAQR5	4439	ovarian cancer	21761364
ATP1A2	4410	tumorogenesis	23474907	PARK2	3729	Parkinson’s D.	26860075
BCL2L15	2650	apoptosis	16690252	PBLD	2077	hepatocarcinoma	26594798
BPNT1	1128	ovarian cancer	20628624	PCGF5	5089	Alzheimer’s D.	16385451
C15orf40	523	uncharacterized		PCSK5	8613	tumorogenesis	21094132
C17orf75	2895	uncharacterized		PDAP1	1926	proliferation	23555679
C17orf75	3672			PDCD4	3221	tumorogenesis	26871813
C21orf58	2668	uncharacterized	11707072	PEX2	3056	cerebellar ataxia	21392394
C4orf19	2068	uncharacterized		PGPEP1	1476	liver cirrhosis	25687677
C6orf170	4113	uncharacterized	20159594	PIK3R2	3345	tumorogenesis	26677064
C8orf44	336**	uncharacterized		PNPLA1	1991	childhood obesity	19390624
C8orf44	1626			PODNL1	1876	uncharacterized	12477932
C9orf85	871	uncharacterized		POFUT1	4679	hepatocarcinoma	27003260
CACNB2	4301	hypertension	25966706	POLH	5550	ovarian cancer	25831546
CACNG8	3218	cardiomyopathy	26710323	PPM1K	2192	diabetes mellitus	23446828
CACNG8	5006			PPP1R12B	5156	childhood asthma	23640410
CACNG8	7535			PRRG4	998	Parkinson’s D	19772629
CALHM1	2896	Alzheimer’s D.	26944452	PSMB2	2925	proteolysis	21660142
CCBE1	3321	ovarian cancer	19935792	PTCD3	4116	osteosarcoma	19427859
CCDC114	261*	dyskinesia	23506398	PTK6	2233	tumorogenesis	27311570
CD109	6841	bladder cancer	20946523	QRFPR	1949	metabolic S.	16648250
CD36	4042	atherosclerosis	16515687	RAB11FIP1	4928	cell transport	26790954
CD68	1398	carcinomas	21113139	RAB3IP	3975	tumorogenesis	12007189
CDAN1	4296	erythropoiesis	19336738		7022
CDHR3	4878	asthma	25848009	RAB7L1	1693	Parkinson’s D.	26914237
CEP68	4394	cervical cancer	17570516	RBBP9	1818	tumorogenesis	21933118
CHST5	2946	colon carcinoma	12107080	RGS3	205*	cardiovascular D.	24375609
CHST6	2979	dystrophy	20539220	RPS6KA6	7136	tumorogenesis	26732474
CHST6	3876			SCN11A	5871	neurophaty	25791876
CIAO1	2416	tumorogenesis	9556563	SEPT11	4033	hepatocarcinoma	20419844
CIAO1	3814			SEPT14	1575	Parkinson’s D	27115672
CLEC19A	1747	lectin	12975309	SGTB	3142	lymphopoesis	2158125
CLTC	7006	pancreatic cancer	23228632	SH3GLB1	4856	prostate cancer	27748942
CORO2A	2227	colon cancer	23490283	SLC15A2	4333	hepatocarcinoma	25965825
COX18	1264	tumorogenesis	20819778	SLC17A5	2389	cardiovascular D	27872510
CPM	2698	renal carcinoma	23172796	SLC26A2	5066	colorectal cancer	23840040
CPM	4996			SLC26A4	4210	hearing loss	27729126
CPT2	2557	sudden death	21641254	SLC28A2	2196	chronic hepatitis C	23195617
CYB5RL	3426	transcription	16344560	SLC7A11	6304	tumorogenesis	26729415
CYP20A1	2539	tumorogenesis	15191668	SLC7A14	8487	breast cancer	20379614
CYP20A1	4709			SNX22	902	liver-disease	21988832
CYP27C1	3823	self-rated health	20707712	SOWAHC	3417	retrotransposon	22234889
CYP2W1	2176	colorectal cancer	22993331	SPATA13	5020	colorectal cancer	17599059
DAP3	1842	breast cancer	22287761	SPATA5	5648	microcephaly	26299366
DCAF10	3305	lung cancer	28336923	SPATS2	3332	breast cancer	20379614
DCAF10	4559			SPN	5287	tumorogenesis	25551301
DCLRE1C	2966	Omenn syndrome	25981738	STAC2	2241	inherited ataxias	16713569
DDOST	1782	hyperglycemia	22305527	SYNJ2BP	1298	breast cancer	19349195
DHODH	1709	melanoma	21430780	SYNJ2BP	4175
DHRS9	1281*	tumorogenesis	26254099	TCEB1	1964	tumorogenesis	23083832
DNAL1	4925	dyskinesia	15845866	TIGD6	3439	uncharacterized
DSCR6	1706	Down syndrome	10814524	TMEM156	1593	uncharacterized
ERBB3	5104	tumorogenesis	26689995	TMEM19	3510	uncharacterized
FADS6	1777	liver disease	21988832,	TMEM213	875	uncharacterized
FAM161A	2785	retinal disease	25749990	TMEM214	1190	uncharacterized
FAM227A	4981	cancer	26759717	TMEM50B	1026	uncharacterized
FAM84B	3626	tumorogenesis	25980316	TMEM56	1243	nicotine dependence	20379614
FBLIM1	2126	breast cancer,	23645746	TMF1	4736	prostate cancer	19330832
FBXL22	1411	cardiomyopathy	24324551	TMOD2	7816	bladder cancer	15095301
FBXO27	1535	leukemia	126433	TNFRSF10A	1621	cancer	27780136
FGD4	7619	cancer	22589722	TNFRSF10D	1532	cancer	26542757
FKBP14	1515	ovarian cancer	27931282	TOP3A	3814	leukaemia	22050635
FKBP14	2129			TPRG1L	1754	uncharacterized
FKBP5	7114	schizophrenia	25522420	TRIM72	1885	ischemia	26790476
FXN	3288	metabolic disease	26717909	TRPM7	8079	neuroblastoma	27402209
GDPD1	1559	phosphodiesterase	18991142	TRPM7	8221	carcinoma	26779625
GEMIN8	2172	neuropathy	16434402	TXNDC15	2460	thrombosis	21642008
GGT6	1956	ovarian cancer	25356737	TYW5	3692	schizophrenia	23974872
GK5	3808	glioblastoma	25936394	UACA	6120	lung cancer	22407486
GK5	6355	glioblastoma	25936394	UACA	6120	thyroid diseases	15358194
GLB1L	2224	phosphatase	21382349	UBIAD1	2881	cancer	23759948
GOLGA3	7240	immune disease	17711851	UBXN2A	1665	colon cancer	24625977
GP2	1877	crohn disease	22891285	UPK1B	1513	cancer	16354592,
GPR65	3309	tumorogenesis	24152439	UQCRB	1269	colorectal cancer	22545919
GPR65	3309	immune diseases	15665078	USP29	2*	protease	10958632
GPR82	2664	uncharacterized		VHL	3764	tumorogenesis	27460078
GPRIN2	6676	schizophrenia	27244233	VHL	3898
GTPBP10	1873	prostate cancer	27409348	VWA2	3366	colon cancer	15580307
H6PD	5754	tumorogenesis	15221007	WDR73	1736	microcephaly	25466283
HM13	1745	glioblastoma	28198167	XIAP	5681	ovarian cancer	26779627
IFIT3	1864	pancreatic cancer	25650658	YAE1D1	1548	oral cancer	23318452
ISY1	686**	uncharacterized		ZBTB24	4842	hepatocarcinoma	27730394
IYD	1658	hypothyroidism.	18765512	ZC3H12D	2812	Acute lung injury	26059755
KIAA1456	2536	colorectal cancer	24743840	ZDHHC20	3390	tumorogenesis	20334580
KIF11	3598	tumorogenesis	28011472	ZFP30	3463	hypertension	19851296
KLHL23	2570	tumorogenesis	23676014	ZNF114	1827	transcription factor	8467795
KPNA1	5711	breast cancer	26052702	ZNF197	3446	thyroid cancer	12682018
KREMEN1	2199	schizophrenia	20153141	ZNF320	5534	glioblastoma	11536051
KREMEN1	2792	schizophrenia	20153141	ZNF429	2081**	transcription factor	7865130
LAX1	2057	uncharacterized		ZNF445	8820	transcription factor	16368201
LILRA6	2201	tumorogenesis	26769854	ZNF461	3087	transcription factor	15004467
LIMD1	5735	breast cancer	27656835	ZNF549	3736	transcription factor	16344560
LIMS1	3931	cancer	27590440	ZNF557	4791	transcription factor	15851553
LMOD3	3224	myopathy	25250574	ZNF626	4620	liver diseases	18255255
LMOD3	3993	Alzheimer’s D	22881374	ZNF667	3240	transcription factor	17397802
METTL6	1188	breast cancer	25151356	ZNF716	2799	cardiovascular D	24376456
MR1	3664	hepatocarcinoma	26823810	ZNF780B	5415	transcription factor	15057824
MREG	1540	pulmonary D	20463177	ZNF84	4920	transcription factor	11856868
				ZNF841	3422	transcription factor	24280104

Notes: * - 5’UTR, **- CDS; others – 3’UTR, D - disease

The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3’UTR (214 sites), CDS (3 sites), and 5’UTR (4 sites). The mRNAs of 27 genes have four binding sites, seven genes have five binding sites, and CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites. The mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these sites are located in the 3’UTRs of mRNAs.

The target genes of the miR-619-5p carry out one or more different functions and are involved in the development of various diseases (Table 1).

The mRNAs of the C17orf75, C8orf44, CIAO1, CPM, CYP20A1, DCAF10, FKBP14, RAB3IP, SYNJ2BP, VHL genes have two complete complementary binding sites for miR-619-5p, and the mRNA of the CACNG8 gene has three such binding sites. This indicates a stronger dependence of the expression of these genes on miR-619-5p.

One of the methods to establish the credibility of the presence of miRNA binding site in the mRNA is to verify this site in the mRNAs of orthologous genes. In finding the miRNA binding sites raises the question of the level of reliability of the found sites. One effective way to establish the credibility of the binding sites is to establish binding sites in the orthologous genes and the identification of orthologous miRNA. Location of binding site in the protein coding region facilitates its conservation in evolution, especially if the corresponding oligopeptide plays an important role in the function of the protein. miR-619-5p binding sites with complete complementarity (ΔG/ΔGm is 100%) to the mRNAs of the four genes are located in the 5’UTRs (Table 2).

Table 2

Variation of positions and nucleotide sequences of miR-619-5p binding sites in the 5’UTRs of mRNAs of mammal genes

Species	Gene	Positionof site, nt	Nucleotide sequence
Hsa	CCDC114	261	GCAUGCUGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Hsa	DHRS9	1281	GCGCGGUGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Hsa	RGS3	205	GCGCAGUGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Ptr	RGS3	1	GCGCAGUGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Nle	RGS3	205	GCACGGUGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Hsa	USP29	2	CUGGCCAGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Pab	USP29	52	CUGGCCAGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Nle	USP29	52	CUGGCCAGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Mle	USP29	47	CUGGCCAGGCUCAUGCCUGUAAUCCCAGCACUUUGG
Can	USP29	98	CUGGCCAGGCUCAUGCCUGUAAUCCCAGCAUUUUGG
Ggo	USP29	100	CUGGCCAGGCUCAUGCCUGUAAUUCCAGCACUUUGG
Rro	USP29	52	CUGGCCAGGCUCAUGCCUGUAAUCGCAGCACUUUGG

Notes: In the table 2-5 the bold type indicates the binding site of miR-619-5p

Table 5

Variation of nucleotide sequences of miR-619-5p binding sites in the 3’UTR of mRNAs of ADAM17, ALDH3A2, and ARL11 of orthologs

Species	Gene	Position, nt	Nucleotide sequence
Hsa	ADAM17	3466	TGGGAGTGGTGGCTCATGCCTGTAATCCCAGCACTTGGAGAGG
Cat	ADAM17	3485	GGGGCGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Mmul	ADAM17	3491	GGGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Mne	ADAM17	3438	GGGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Ptr	ADAM17	3449	TGGGAGTGGTGGCTCATGCCTGTAATCCCAGCACTTGGAGAGG
Rro	ADAM17	3425	GGGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Hsa	ALDH3A2	2617	CGGGCGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Cja	ALDH3A2	3444	CGGGCGTGGTGGCTCATGCCTGTAATCCCAGCACTTTAGGAGG
Ggo	ALDH3A2	2712	CGGGCGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Mmul	ALDH3A2	2509	CGGACATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Mne	ALDH3A2	2504	CGGACATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Nle	ALDH3A2	2714	TGGTCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Pab	ALDH3A2	2297	TGGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Ppa	ALDH3A2	2715	CGGGCATGGTGGCTCATGTCTGTAATCCCAGCACTTTGGGAGG
Ptr	ALDH3A2	2711	CGGGCATGGTGGCTCATGTCTGTAATCCCAGCACTTTGGGAGG
Rro	ALDH3A2	2727	CGGACGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGG
Hsa	ARL11	1033	TTGGCCCGGTGGCTCATGCCTGTAATCCCAGCACTGTGGGAGA
Cat	ARL11	1642	CAGATGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGTGG
Mfa	ARL11	1698	CAGATGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGTGG
Mmul	ARL11	1747	CAGATGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGTGG
Mne	ARL11	1024	TTGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGA
Mne	ARL11	1471	CAGATGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGTGG
Ptr	ARL11	1353	CGGGCATGGTGGCTCATGTCTGTAATCCCAGCACTTTGGGAGG
Rro	ARL11	1254	CAGGTGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGCGG

Before the 5’ end and after the 3’ end of miR-619-5p binding site, nucleotides are not homologous. The mRNAs of RGS3 and USP29 orthologous genes have binding sites in H. sapiens, N. leucogenys, P. abelii, M. leucophaeus, C. angolensis palliatus, G. gorilla, and R. roxellana.

miR-619-5p has two binding sites in the 5’UTRs of mRNAs of ANAPC16, CYB5D2, and PRR5 and three binding sites in the mRNA of DNASE1.

mRNAs of some genes have binding sites for miR-619-5p within their 5’UTRs and 3’UTRs or CDSs and 3’UTRs. For example, ATAD3C, C14orf182, and CYB5RL have miR-619-5p binding sites in the 5’UTRs and 3’UTRs, and C8orf44, ISY1, and ZNF714 have miR-619-5p binding sites in the CDSs and 3’UTRs.

The nucleotide sequences of miR-619-5p binding sites are located in the CDSs of the C8orf44, C8H8orf44, ISY1, ZNF429, and ZNF714 genes and encode the following oligopeptides (Table 3). C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is also present in the orthologous proteins of P. abelii, P. anubis, P. paniscus, and P. troglodytes. The mRNA of transcription factor ZNF429 and ZNF429 genes binding sites are encoded the AHACNP oligopeptide in the another reading frame. The first two oligopeptides are encoded in one open reading frame (ORF) and the amino acid sequences are highly conserved. The homologous oligonucleotide of the miR-619-5p binding site in the mRNA of ZNF714 gene codes for an oligopeptide in a different ORF.

Table 3

Variation of amino acid sequences coding in miR-619-5p binding sites in the mRNAs of orthologous genes

Species	Gene	Amino acid sequence
Hsa	C8orf44	HWKGRARWLMPVIPALWEAKA
Hsa	C8H8orf44	HWKGRARWLMPVIPALWEAKA
Pab	C8H8orf44	HWKGWARWLTPVIPALWEAKA
Pan	C8H8orf44	HWKGRARWLMPAIPALWEAKX
Ppa	C8H8orf44	HWKGRAQWLTPVIPALWEAKA
Ptr	C8H8orf44	HWKGRAQWLTPVIPALWEAKA
Hsa	ISY1	EKERQVRWLMPVIPALWEAEA
Hsa	ZNF714	KIQQGMVAHACNPNTLRGLGE
Ggo	ZNF714	KIQQGMVAHACNPNTLRGLGE
Ptr	ZNF714	KIQQGMVAHACNPNTXRGLGE
Ppa	ZNF714	KIQQGMVAHACNPNTLRGLGE
Hsa	ZNF429	IHRMGVVAHACNPSTLGGRGG
Mfa	ZNF429	IHRLGVVAHACNPSTLGGRGG
Mmu	ZNF429	IHRLGVVAHACNPSTLGGRGG
Mne	ZNF429	IHRLGVVAHACNPSTLGGRGG

The presence of miR-619-5p binding sites in the CDSs of five genes with different functions and the evolutionary conservation of these sites signify the role of miRNA in the regulation of the expression of these genes. The nucleotide sequences of specific regions of mRNAs of C8H8orf44, C8orf44, ISY1, ZNF429, and ZNF714 genes that contain miR-619-5p binding sites in the CDSs are homologous among themselves and to the binding sites located in the 5’UTRs and 3’UTRs.

The miRNA binding sites in the coding region, as opposed to the 3’UTR and 5’UTR, clearly demonstrate the relationship between miRNA and mRNA by their conserved amino acid sequences in orthologous proteins. miRNA binding site can be translated by two open reading frames that encode WLTPVIPA and AHACNPS oligopeptides. In the third reading frame, the miR-619-5p binding site has a stop codon. However, in the genes studied, no such sequence was found. In the absence of complete complementarity between miR-619-5p and its binding site, miR-619-5p uses a site containing the corresponding mutation in the CDS for the regulation of gene expression. Thus, a single miRNA binding site in the mRNA of various genes may correspond to three different oligopeptides. Generally, one out of these three oligopeptides is present in the proteins encoded by the orthologous genes.

ISY1 orthologous genes in H. sapiens, P. troglodytes, and N. leucogenys encode a protein containing QVRWLMPVIPALWEAEAGGSQA oligopeptide sequence (Table 4).

Table 4

Amino acid sequences coding in miR-619-5p binding sites in the mRNA of ISY1 gene of orthologous genes

Species	Amino acid sequence
Hsa	PGVRELFEKERQVRWLMPVIPALWEAEAGGSQALPPPRKTRAELMKA
Ptr	PGVRELFEKERQVRWLMPVIPALWEAEAGGSQALPPPRKTRAELMKA
Nle	PGVRELFEKERQARWLTPVIPALWEAEAGGSQALPPPRKTRAELMKA
Hsa*	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Bmu	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Cdr	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Cfa	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Cja	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Eca	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Ggg	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Mmu	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Nle	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Oar	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Pab	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Ppa	PGVRELFEKEP----------------------LPPPRKTRAELMKA
Rro	PGVRELFEKEP----------------------LPPPRKTRAELMKA

* RAB43 - human ISY1 paralog gene

However, the RAB43 gene, which is paralogous to human ISY1, lacks the nucleotide sequence encoding the QVRWLMPVIPALWEAEAGGSQA oligopeptide. Additionally, ISY1 gene in the genomes of other animals also lacks the nucleotide sequence encoding this oligopeptide (Table 4).

Nucleotide sequences of miR-619-5p binding sites in the mRNAs of ADAM17, ALDH3A2, and ARL11 orthologous genes are shown in Table 5.

These orthologous genes are characterized by highly conserved nucleotide sequence GGCTCATGCCTGTAATCCCAGC of miR-619-5p binding sites. This shows that the interaction of miR-619-5p with mRNAs of these genes is conserved during evolution. Some of the human miR-619-5p target genes and their corresponding orthologous genes have two miR-619-5p binding sites in their mRNAs.

Table 6 shows the nucleotide sequences of two miR-619-5p binding sites in the 3’UTR of mRNAs of ERBB3, FBLIM1, and FKBP14 orthologous genes.

Table 6

Variation of nucleotide sequences of two miR-619-5p binding sites in the 3’UTR of mRNAs of ERBB3, FBLIM1, and FKBP14 of orthologs

Species	Gene	Position, nt	Nucleotide sequence
Hsa	ERBB3	4950	CGGGCATGGTGGCTCATGCCTGTAATCTCAGCACTTTGGGAG
Hsa	ERBB3	5104	TGGGTGCAGTGGCTCATGCCTGTAATCCCAGCCAGCACTTTG
Csa	ERBB3	4989	CGGGCATGGTGGCTCATGCCTGTAATCCTAGCACTTTGGGAG
Csa	ERBB3	5149	TGGGCGCTGTGGCTCATGCCTGCAATCCCAGCACTTTGGGAG
Mfa	ERBB3	5114	TGGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAG
Mfa	ERBB3	5269	TGGGCGCTGTGGCTCATGCCTGCAATCCCAGCCCTTTGGGAG
Mmu	ERBB3	5114	TGGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAG
Mmu	ERBB3	5269	TGGGCGCTGTGGCTCATGCCTGCAATCCCAGCCCTTTGGGAG
Mne	ERBB3	5112	CGGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAG
Mne	ERBB3	5267	TGGGCGCTGTGGCTCATGCCTGCAATCCCAGCCCTTTGGGAG
Pan	ERBB3	5106	CGGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAG
Pan	ERBB3	5274	TGGGCGCTGTGGCTCATGCCTGCAGTCCCAGCACTTTGGGAG
Ptr	ERBB3	5105	CGGGCATGGTGGCTCATGCCTGTAATCTCAGCACTTTGGGAG
Ptr	ERBB3	5243	TGGGTGCAGTGGCTCATGCCTGTAATCCCAGCCAGCACTTTG
Mne	FBLIM1	1938	TGGGCGTGGTGGCTCATGCCTGTAATCCCTGCACTTTGGGAG
Mne	FBLIM1	5267	TGGGCGCTGTGGCTCATGCCTGCAATCCCAGCCCTTTGGGAG
Pab	FKBP14	1514	CAGGCACGGTGGCTCACGCCTGTAATCCCAGCACTTCGGGAG
Pab	FKBP14	2128	TGGGTGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGGG

Notes: The black type indicates the binding site of miR-619-5p

Table 7 shows the degree of conservation of miR-619-5p binding sites in the 201 mRNAs of target genes. All mRNAs with complete complementarity to miR-619-5p binding sites (ΔG/ΔGm is 100%) were divided into four groups, and the frequency of occurrence of nucleotides was determined in each group. The results suggest that miR-619-5p binding sites are highly conserved. The binding site GGCTCATGCCTGTAATCCCAGC does not change and in each of the four gene groups the observed variability of nucleotides on the right and left is high.

Table 7

Variation of nucleotide sequences of mRNA region with miR-619-5p binding sites (See Additional file 1, 2, 3 and 4)

Discussion

Here we have identified many miRNAs binding sites in the mRNAs of 201 human genes which indicates that umiRNAs act as coordinators of gene expression by participating in many biological processes. Previous studies have shown the influences of miRNAs on the expression of genes that encode the transcription factors [19, 20] and on the expression of proteins that participate in the cellular cycle [3, 21–23], apoptosis [4, 24–26], and stress responses [27]. It was shown the role of the mir-619-5p in the regulation of different pathological processes [28]. It was investigated the correlations between the expression of MALAT1 and miR-619-5p, in addition to the association between the clinicopathological features and survival outcomes of patients with stage II and III colorectal cancer tumors [28]. It was observed, that hsa-miR-619-5p and hsa-miR-1184 microRNA expression significantly increased in prostatic cancer. MicroRNA-gene-net analysis indicated that miR-619-5p and other some miRNAs had the most important and extensive regulatory function for Qi-stagnation syndromes and Qi-deficiency syndromes in coronary heart disease [29].

One or several umiRNAs regulating the expression of hundreds of genes can create a system of interconnected processes in cells and organisms. Such role of these umiRNAs is possible because they circulate in the blood and have access to nearly all cells of an organism [30-32]. Our results provide the basis for studying the systemic roles of unique and normal miRNAs in the regulation of gene expression in human cells. The expression of many target genes is regulated by umiRNAs does not allow individual mRNAs of target genes to be expressed in more degree than others. The greater expression of one mRNA, the larger number of umiRNAs bind to this mRNA. This allows one umiRNA to maintain a certain balance of expression of the corresponding target genes. If umiRNA expression changes, such system is vulnerable. This will cause the development of pathology in the cell, tissue or body.

Conclusions

The majority of miR-619-5p binding sites are located in the 3’UTRs of mRNAs of target genes. Some genes have miRNA binding sites in the 5’UTRs of mRNAs. It is necessary to maintain nucleotide sequences of the binding site of umiRNA in the CDSs of several genes. Different genes have binding sites for miRNAs that are read in different open reading frames. Therefore, identical nucleotide sequences encode different amino acids in different proteins. In encoded proteins, these sites encode conservative oligopeptides. The binding sites of miR-619-5p in 3’UTRs, 5’UTRs and CDSs are conservative in the orthologous mammalian genes.

Variation of nucleotide sequences of mRNA region with miR-619-5p binding sites of genes from CSL6 to COX18 (Conservative binding sites are in bold) (PDF 218 kb)

Variation of nucleotide sequences of mRNA region with miR-619-5p binding sites of genes from GK5 to HM13 (Conservative binding sites are in bold) (PDF 106 kb)

Variation of nucleotide sequences of mRNA region with miR-619-5p binding sites of genes from IFIT3 to SLC26A4 (Conservative binding sites are in bold) (PDF 139 kb)

Variation of nucleotide sequences of mRNA region with miR-619-5p binding sites of genes from LC28A2 to ZNF841 (Conservative binding sites are in bold). The data given in the Additional files 1, 2, 3 and 4 demonstrate the variability of the nucleotides before and after the binding sites of miR-619-5p, which is shown in the Weblogo schemes in the table 8. (PDF 151 kb)