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Characteristics of miRNA binding sites in mRNAS of human and mouse titin gene

: We have studied characteristics of miRNA (microRNAs) binding sites in mRNAs (matrix RNAs) of human, primate and mouse titin gene. miRNAs are small non-coding RNAs with the length about 21-22 nucleotides binding with mRNAs of genes and blocking or disturbing their translation. Titin is the largest protein of heart muscle tissue that is a base of myofibril. Defects of titin synthesis lead to malfunction of muscle tissue, for example, to the heart failure which is one of the widest reasons of the death in the world. We have found differences and similarities of characteristics of miRNA binding sites in human and mouse titin gene mRNAs. The differences are the following: different number of binding sites, different values of binding energy and different nucleotide sequences of orthologous human and mouse miRNAs. The similarities are concluded in that all of these sites are located in protein-coding part of mRNA and they all have particular complementarity. But changing some nucleotides can help to get artificial miRNAs with ideal complementarity and maximal effect on expression. We have noticed that characteristics of miRNA binding sites in mRNAs of titin gene between different species of primates are more similar than between human and mouse. It can be explained by different evolutionary distance between these species. So the model of miRNA regulation of mouse titin synthesis is not completely adequate for human titin gene, but weakness of miRNA interaction with mRNA of mouse titin gene can be compensated by increasing of miRNA concentration in relation to mRNA.


Introduction
Titin is a protein of muscle tissue. It is the largest protein in the nature and plays enormous role in providing elasticity and structural integrity of sarcomers [1]. For example, the longest transcript variant of human titin gene encodes 35991 amino acids and includes all 363 exons of this gene. Disturbance of titin synthesis causes the development of serious cardiovascular diseases, such as dilated cardiomyopathy, heart failure, ischemic heart disease, myocardial infarction and etc. Different titin isoforms are synthesized in various types of muscle tissue (heart, smooth and skeletal striated muscle tissue) and are encoded by different combinations of exons [2].
Recently miRNA binding with mRNAs of different genes, participating in the development of cardiovascular diseases, was studied [3][4]. For example, it was proved, that high level of miR-208b expression leads to cardiac hypertrophy in titin-based dilated cardiomyopathy [5]. But characteristics of miRNA interactions with mRNAs of titin gene were not studied. So it was important to establish what kind of miRNAs bind with mRNAs of titin gene? Since miRNA binding sites in mRNAs of orthologous genes can differ it is necessary to study characteristics of miRNA binding with mRNAs of orthologous genes, especially with mRNAs of human and mouse titin gene because mouse is used in experimental research. So it was important to compare characteristics of miRNA binding sites in mRNAs of titin gene. It is possible that differences can be observed in miRNA binding with mRNAs of different titin isoforms. ncbi.nlm.nih.gov/genbank/). Nucleotide sequences of 2568 miRNAs were taken from the miRBase (www. mirbase.org/) and 3707 other miRNAs were discovered in 2015 by Eric Londina and other scientists [6]. Human miRNAs have abbreviation "hsa" (Homo sapiens) in the beginning of their names. Mouse miRNAs have abbreviation "mmu" (Mus musculus). Free energy of miRNA-mRNA binding (ΔG), ΔG/ ΔG m ratio (%), positions and schemes of potential miRNA binding sites were calculated by the program MirTarget [7][8]. ΔG m (maximal ΔG) is free energy of miRNA binding with nucleotide sequence that is absolute complementary to this miRNA. ΔG/ΔG m ratio was used as comparative criterion of miRNA-mRNA interaction. ΔG/ΔG m ratio should be equal or more than 85% because 15% of ΔG m correspond to three nucleotides of miRNA sequence which can encode one amino acid. Binding site with value of ΔG/ΔG m ratio, that is less than 85%, is counted as unreliable because it has low specifity and can bind different miRNAs. Diagrams of evolutionary conservation of olygopeptides encoding by miRNA binding sites were created by the program WebLogo (https:// www.weblogo.berkeley.edu/). The information about microRNA expression was taken from Human miRNA tissue atlas (https://ccb-web.cs.uni-saarland. de/tissueatlas) and TiGER: Tissue-specific Gene Expression and Regulation (bioinfo.wilmer.jhu.edu/ tiger/). Positions of miRNA binding sites were compared with single nucleotide polymorphisms (SNPs) of human titin gene (https://www.ncbi.nlm.nih.gov/ snp/?term=TTN).
Four mouse miRNAs (mmu-miR-34a-3p, mmu-miR-136-3p, mmu-miR-374c-3p and mmu-miR-494-5p) are orthologous to corresponding human miRNAs (Table «b»). Differences of mouse miRNA nucleotide sequences and corresponding human sequences are one-three nucleotides. For example, the sequence of hsa-miR-34a-3p has a cytosine (C) in the beginning that is absent in the sequence of mmu-miR-34a-3p. So the length of hsa-miR-34a-3p is 22 nucleotides while the length of mmu-miR-34a-3p is 21 nucleotides. The sequence of hsa-miR-136-3p has nucleotide C in the beginning that is absent in the sequence of mmu-miR-136-3p. But it hasn't an uracil (U) that is present in the sequence of mmu-miR-136-3p. So the length of these miRNAs is the same. hsa-miR-374c-3p is three nucleotides longer (22 nucleotides) than mmu-miR-374c-3p (19 nucleotides) because hsa-miR-374c-3p has nucleotide C in the beginning and nucleotides A (adenine) and U at the end that are absent in the sequence of mmu-miR-374c-3p. hsa-miR-494-5p has nucleotide U at the end of sequence that makes it longer than mmu-miR-494-5p. The length of hsa-miR-494-5p is 23 nucleotides and the length of mmu-miR-494-5p is 22 nucleotides.
Than we have calculated binding of 18 human miRNAs and mmu-miR-34a-3p, mmu-miR-136-3p, mmu-miR-374c-3p, mmu-miR-494-5p with mRNA of mouse titin gene. Only eight miRNAs bind with mRNA of mouse titin gene with values of ΔG/ΔG m equal 85 % and more (hsa-miR-19-36945-3p, hsa-miR-11-28905-3p, mmu-miR-34a-3p, hsa-miR-1278, hsa-miR-544b, hsa-miR-34a-3p, hsa-miR-4693-5p and hsa-miR-4495) ( Table «с These miRNAs have nine binding sites. Only hsa-miR-19-36945-3p bind with mRNA of mouse titin gene with value of ΔG/ΔG m equal to 90%. hsa-miR-4693-5p has the lowest value of ΔG/ΔG m ratio that is equal to 85%. It has binding site in the 282 nd exon of mouse titin gene. hsa-miR-19-36945-3p, hsa-miR-11-28905-3p, hsa-miR-544b and hsa-miR-34a-3p have values of miRNA-mRNA interaction energy equal to -100 kJ/mole and lower. Other miR-NAs have higher values of binding energy than these miRNAs. hsa-miR-11-28905-3p has two binding sites in the 58 th exon with the lowest level of interaction energy (-113 kJ/mole) and ΔG/ΔGm ratio that is equal 86%. hsa-miR-4495 has the highest level of miRNA-mRNA interaction energy that is equal -91 kJ/mole. Binding site of this miRNA is located in the 295 th exon of mouse titin gene. The single mouse miRNA binding site in mRNA of mouse titin gene is binding site of mmu-miR-34a-3p that is located in the 66 th exon. It has value of ΔG/ΔG m that is equal 86 %. Binding site of hsa-miR-34a-3p is located very far from binding site of mmu-miR-34a-3p in the mRNA of mouse titin, in the 93 rd exon, and has value of ΔG/ ΔG m that is equal 87 %. hsa-miR-1278 has binding site in the 83 rd exon of mouse titin gene and value of ΔG/ΔG m that is equal 88 %. hsa-miR-544b has binding site in the 87 th exon of mouse titin gene and value of ΔG/ΔG m that is equal 89 %.
One of the evidences of miRNA binding site existence is presence of it in mRNAs of orthologous genes. We have studied binding of 6271 human miR-NAs with mRNAs of titin gene of 14 species of primates (Homo sapiens, Pan troglodytes, Pongo abelii, Macaca fascicularis, Papio anubis, Pan paniscus, Colobus angolensis, Chlorocebus sabaeus, Rhinopithecus roxellana, Callithrix jacchus, Aotus nancymaae, Saimiri boliviensis, Gorilla gorilla, Nomascus leucogenys). It was found that only hsa-miR-494-5p and hsa-miR-578 bind with mRNAs of primate titin gene with values of ΔG/ΔG m equal to 90% and more. Although orthologous miRNAs of various primate species are different, primates have conservative genes of miRNA synthesis and conservative miRNA binding sites. For example, Figure1: binding sites of hsa-miR-494-5p in titin mRNAs of primates encode conservative heptapeptide RETTLTT, in which the last aminoacid changed (threonin is changed to alanin or serin). Figure2: binding sites of hsa-miR-578 in titin mRNAs of primates encode conservative heptapeptide TVPGAQE. As we can see, flanking amino acid sequences are even more conservative than these olygopeptides themselves. Conservation of olygopeptides, encoding by miRNA binding sites, proves conservation of these mRNA regions. Characteristics of miRNA binding sites in mRNAs of primate titin gene are more similar than in mRNAs of human and mouse gene because of different evolutionary distance between these species. miRNAs can't effectively block translation of titin because they are not absolutely complementary to their sites. But artificial miRNAs, that would be absolutely complementary to these sites and have ΔG/ΔG m ratio equal 100 %, could effectively bind with mRNAs of titin genes and block their translation (Table «d»). There are 25 miRNA binding sites in this table that have ΔG/ΔGm ratio equal 90% and more. Nucleotides of miRNAs, decreasing energy of miRNA-mRNA interaction, are signed by red color. For example, we need to change three nucleotides in the sequence of hsa-miR-374b-3p binding site that is located in the position 17241 of human titin gene to synthesize absolutely complementary artificial miR-NA to this site. In other case, we need to change only one nucleotide in the sequence of mmu-miR-494-5p binding site located in position 1302 to get ideal artificial miRNA. In the case of hsa-miR-494-5p binding site, located in position 1301 of human titin gene, two nucleotides are needed for synthesis of complementary sequence for this site. In relation to mouse titin gene mRNA, it is needed to change two nucleotides in the sequence of hsa-miR-19-36945-3p located in position 30584 to get artificial miRNA.

Conclusions
We have found differences and similarities of characteristics of miRNA binding sites in mRNAs of human and mouse titin gene. Differences are the following: 1. Human titin mRNA has 23 miRNA binding sites whereas mouse titin mRNA has only nine sites.
2. 19 miRNAs (18 human miRNAs and one mouse miRNA) interact with mRNA of human titin but only eight miRNAs from this number bind with mRNA of mouse titin gene.
3. ΔG/ΔG m ratio of miRNA binding sites in the mRNA of human titin gene varies from 89% to 94% but in the mRNA of mouse titin gene it varies from 85% to 90%. 4. Orthologous human and mouse miRNAs have different nucleotides in their sequences.
Similarities of characteristics of miRNA binding sites in human and mouse titin mRNA are the following: 1. Positions of miRNA binding sites in the mRNA of mouse titin gene are very close to such positions in the mRNA of human titin.
2. All miRNA binding sites are located in protein-coding part of these mRNAs.
Thus characteristics of miRNA binding sites in mRNAs of human and mouse titin gene are different and it should be taken into account. So the model of mouse titin synthesis regulation by miRNAs is not completely adequate for human titin gene. Nevertheless, increasing of miRNA concentrations in relation to mRNA of mouse titin gene can cause the same effect on expression of this gene as in human. In something's totality, obtained results permit to suppose that expression of titin gene is weekly regulated by miRNAs in human and mouse organisms.