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O.A.Orlovsky
Ph.D (Biol), D.Sc(MA), staff scientist, R.E.Kavetsky Institute of experimental pathology, oncology and radiobiology
V.O. Shlyakhovenko Doctor of medicine, professor, principal staff scientist, the same place of employment O.A.Samoylenko Principal engineer, the same place of employment
TOWARDS THE INFORMATIONAL REDUNDANCY OF C-MYC- AND NF-KB-p50-TFBS-COMPLEMENTARY MOTIFS IN miRNA
Summary
Aim. Characterization of the overlapping TFBS-complementary motifs in miRNA as the signs of the informational redundancy miRNA-mediated transcription control.
Methods. Computer search for the objects of interest in the reference mature miRNA sequences given in miRBase - the main international miRNA database being supported by Manchester University.
Results. Only 2 c-myc/c-myc intrafactor overlaps were found and only in the Homo sapiens miRNA set. P50/p50 intrafactor overlaps were found in all miRNA sets have been studied: 25 (10 of them - double) in Homo sapiens, 20 (6 - double) in Mus musculus and 5 (without double) - in Rattus norvegicus. C-myc/p50 (interfactor) overlaps were found in 7 miRNAs of the Homo sapiens set, 1 - in Mus musculus and 1- in Rattus norvegicus. All these overlaps were characterized by the number of the overlapped nucleotides and by 3'-end or 5'-end position of the overlapped motifs to one another. A hypothetical mechanism was suggested to optimize the informational redundancy level in the biological systems. In accordance to this hypothesis, the ligand-receptor interactions, in their state space, may be described by the trigonometric polynomials, and each chain of such interactions - as multiply differentiating superposition of these polynomials.
Conclusions. Informational redundancy of miRNA-mediated transcription control is higher in the evolutionary more progressive systems and may be optimized with the internal biophysical mechanisms
Keywords: microRNA, transcription control, informational redundancy.
Introduction
In all kinds of the complex systems, especially in the biological ones, different types and levels of the informational redundancy secure multilevel regulation stability [1]. At the same time, redundancy in an evolving and/or training system must not be absolute, otherwise the stage selection in this system becomes impossible [2, 3]. In this paper, we shall view some aspects of this balance.
In our previous paper [4] we have demonstrated that many of the miRNAs contain one or more TFBS-complementary motifs, both conventional and modi-
fied, and thus are potentially able to serve as alternative transcription factors. In addition, we have noted the overlaps of the TFBS-complementary motifs and interpreted them as a sign of informational redundancy in transcription control.
Supposing the TFBS and TFBS-complementary motifs overlaps and informational redundancy are the subjects of a special interest, we have analysed them more thoroughtly in the present study, having as a goal deeper understanding of informational flows in transcription control and signal transduction in whole.
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Materials and methods
All main preparative procedures were described in our previous paper [4]. Namely:
Nucleotide sequences of the mature miRNAs were retrieved in the miRBase — the officially main miRNA database in the world being supported by Manchester University (UK) [5] - and saved in the Microsoft Word format (.doc or .docx).
C-myc-TFBS-complementary and NF-KB-p50-TFBS-complementary motifs were determined as the short RNA nucleotide sequences, complementary to the c-myc-binding and NF-KB-p50-binding DNA motifs described in our previous paper [6] and were found in the miRNA sequences by the same way as it was described in [6] for the DNA TFBS motifs. Overlaps of the TFBS-complementary motifs were specially noted as a sign of informational redundancy of miRNA-mediated transcription control.
Note 1. We name the TFBS variations as "deletions", "insertions" or "exchanges", i.e., such as mutations are named commonly. But we can't know, are they real mutations or constitutional variations. Indeed, we can establish a true mutation only if we know that certain nucleotide sequence was previously without the variation we observe now and only later got it. Moreover, some our finds (see below) suggest that most of the TFBS and TFBS-complementary site variations are just constitutional, not true mutant.
Overlaps analysis (a new procedure in comparison with [4]) was performed using the Supplementary materials of the [4] paper and a new form in which the list of all possible c-myc-TFBS-complementary motifs was put over the list of NF-KB-p50- TFBS-complementary motifs. In the resulting Tables 1 and 2
we have noted the data in the following standard order: we have supposed (only for standardization, not more!) that a shorter motif overlaps a longer one and noted how many nucleotides and from what end (3' or 5') of the longer motif were overlaped with the shorter one. If the overlapped motifs were of identical length (only 1 case, see Table 1), we have noted this as "N3 =N5'".
Statistical evaluations. If it was necessary, significance of the differences was calculated using the exact Fisher's method. But, since such factorials of big numbers as, for example 435! or 2558!, in practice may be used only in the rough, in ax10n form, where a is a rational number [7], the final results where also evaluated approximately, as P<0.001, etc.
Results and discussion
This section is naturally divided into two subsections describing: 1) immediately observed quantitative regularities and 2) view on the biological meaning of them.
I. Quantitative regularities
The integrated search results are given in the Tables 1and 2. Before a reader will look them, we must give one explanation.
In the cases of the double overlaps, such notations as, for example, 3*2=6 are given in the 4-th bar of the Table 1. This means the following. The first number: total number of the double overlaps of certain kind (del/conv/ins in this case) in this species miRNA set is equal to 3; the second number: each member of each double overlap is overlapped with 2 other members; the 3-rd number: total number of the individual overlaps in the double-overlapped structures of this kind in this species miRNA set is equal to 6.
Table 1.
The intrafactor (c-myc/c-myc and p50/p50) overlaps of c-myc-TFBS-complementary and NF-KB-p50-TFBS-complementary motifs in the miRNA of Homo sapiens, Mus musculus and Rattus norvegicus_
Species Kind of an overlap, miRNA name Number and location of the overlapped nucleotides Number of overlaps of certain kind E overlaps (of them -double)
C-myc/c-myc overlaps
Homo sapiens, commun miR signature - hsa Ins/del, miR-23c 5(5') 1 2 (0) of total 425 sites
Del/ins, miR-126 5(3') 1
Mus musculus, commun miR signature - mmu Absent 0 0 0 of total 256 sites
Rattus norvegicus, commun miR signature - rno Absent 0 0 0 of total 150 sites
p50/p50 overlaps
Del/conv/ins, miR-593, 671, 4253 9(3') + 10(3') 3x2=6
Del/conv/del, miR-611 9(3') + 9(5') 1x2=2
Exch/ins,
y
miR-623, 1343, 4257, 4278 10(3') 4
Exch/del, miR-1257, 7851 9(5') 2 25*(10**) of total 132 sites
Homo sapiens, commun miR signature - hsa Del/conv, miR-3117, 3689b, 4287, 6728, 6737 9(3') 5
Conv/del, miR-3689d 9(5') 1
Ins/exch, miR-4488 10(5') 1
Del/exch, miR-4713 9(3') 1
Conv/del/exch, miR-6503 9(5') + 7(5') + 7(3') 1x2=2
Conv/ins, miR-6785 10(3') 1
Ins/conv, miR-29a, 133c 10(5') 2
Exch/ins, miR-185, 3085, 7009 10(3') 3
Del/conv/ins, miR-671 9(3') + 10(3') 1x2=2
Del/conv, miR-8108 9(3') 2 20*(6**)& of total 77 sites
Exch/del, miR-760, 3072, 6990, 8100 9(5') 4
Mus musculus, commun miR signature - mmu Conv/del, miR-1895, 3110 9(5') 2
Conv/del/del, miR-3059 9(3') + 8(5') 1x2=2
Exch/exch miR-3471 9(3') = 9(5') 1
Conv/ins, miR-6898 10(3') 1
Ins/exch/del, miR-6964 10(5') + 9(5') 1x2=2
Rattus norvegicus, commun miR signature - rno Exch/ins, miR-185, 3085 9(3') 2 5*(0**) of total 31 sites
Del/conv, miR-671 9(3') 1
Exch/del, miR-760, 3072 9(5') 2
Notes: * - P<0.05 versus the corresponding cell of the c-myc/c-myc part of this table; ** - P<0.01 versus each of two other species miRNA sets. & - The value in the analogous cell in [1, Table 2] is a mistake,
in the present table we give the correct value. But the mistake is enough small to remain statistical significance.
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Table 2.
The interfactor (c-myc/p50) overlaps of c-myc-TFBS-complementary and NF-KB-p50-TFBS-
complementary motifs in the miRNA of Homo sapiens, Mus musculus and Rattus norvégiens
Species Kind of an overlapped c-myc/p50-TFBS-complementary sites, miRNA name Number and location of the overlapped nucleotides Number of overlaps of certain kind E overlaps (of them -double)
Exch/del, miR-134 1(5')/1(3') 1
Homo sapiens, commun miR signature - hsa Del/del, miR-448 1(3')/1(5') 1
Del/ins, miR-519d, 520a, 521-1, 526b 1(5')/ 1(3') 4* 7 (0)
Exch/conv, miR-938 1(5')/1(3') 1
Mus musculus, commun miR signature - mmu Exch/del, miR-134 1(5')/ 1(3') 1 1 (0)
Rattus norvegicus, commun miR signature - rno Exch/del, miR-134 1(5')/ 1(3') 1 1 (0)
Note: * - P<0.01 versus each of others c-myc/p50 overlaps.
In the Table 1, one can see the following significant facts.
x c-myc/c-myc overlaps were found only twice and only in the Homo sapiens miRNA set; double overlaps not found among them; only the sites with insertions and deletions participate in these overlaps;
x p50/p50 overlaps were found multiply in each species miRNA and this difference from the c-myc/c-myc overlaps is statistically significant;
x p50/p50/p50 double overlaps were found in the Homo sapiens and Mus musculus miRNA sets but not in the Rattus norvegicus one, and all interspecies differences in their frequency are statistically significant, with a maximum in the Homo sapiens miRNA set;
x all kinds of the p50-TFBS-complementary motifs (sites) participate in both single and double p50/p50 overlaps; at the same time, some of the overlap types were found much more frequently than others (for example, in the Homo sapiens miRNA set, del/conv overlap was found for 5 times but ins/exch overlap - only 1 time, etc.;
x in overwhelming majority of the overlaps, both single and double and both p50/p50 and c-myc/c-myc, the motifs of different kinds and different length (for example, conv/ins or del/exch) are presented; only in a single case, in the Mus musculus mmu-miR-3471, there was found an overlap between identical motifs (exch/exch);
x registering the overlaps in a standard way -"the shorter motif overlaps the longer one" - we can calculate a ratio of 3'- and 5'-overlaps number; this ratio was found to be strongly dependent on species and amounted: 2.7 for Homo sapiens, 1.5 for Rattus
norvegicus and 0.8 for Mus musculus.
Involving the Table 2 and Supplemented materials into our analysis, we must note the followings.
x If 2 or 3 of p50-TFBS-complementary sites are overlapped between one another, any c-myc-TFBS-complementary site is overlapped with any of them (Supplementary materials);
x the intrafactor overlaps (Table 1) include 5 nucleotides (c-myc/c-myc) or 7 - 10 nucleotides (p50/p50), while the interfactor overlaps (c-myc/p50, Table 2) always include only 1 nucleotide; it is of a special interest that the same relations were found between the corresponding TFBS kinds in the human and mouse DNA (Supplemented materials);
x the interfactor overlaps were found only in 7 miRNAs, and in 6/7 of these cases a c-myc-TFBS-complementary site overlaps the 3'-end single nucleotide of a p50-TFBS-complementary site;
x as well as in the case of the interfactor overlaps, one of the overlap types (namely, deleted c-myc site with inserted p50 site) was found much more frequently (in 4/7 cases) than others (1/7 for each). All these observations point at the followings.
1. They make additional confirmation of a hypothesis about specific function of each kind of the TFBS in DNA and TFBS-complementary sites in miRNA with respect to the transcription control have been suggested in [4, 6] papers. This functional differentiation and switch mechanisms between the modalities are, probably, much more advanced in NF-KB-dependent transcription control than in c-myc-dependent one.
2. They set one to thinking on a special mechanism being able to switch transcription control
u
from c-myc transcription factor to NF-kB or back.
3. They make additional confirmation of a hypothesis on much more young evolutionary age and multiple times higher informational redundancy of NF-kB-dependent transcription control than in c-myc-dependent one, have been suggested in the [4] paper.
II. Transcription control and trigonometric polynomials
Our paper series [4, 6 and the present paper] introduces one new class of the transcription control targets (TFBS located not in a promoter but in a structural gene) and one new class of the transcription controlling agents (miRNAs with their TFBS-complementary motifs). In addition, in these papers we adduce the essential reasons to interpret the modified types of the TFBS and TFBS-complementary motifs not as the accidental variations but as the specialized functional elements. Thereby, this series strongly enlarges the known transcription control system both quantitatively and qualitatively. Especially, in our interpretation, the competitive relations between the transcription control targets (equal but differently located TFBS) and agents (traditional protein transcription factors and miRNAs with the corresponding TFBS-complementary motifs) acquire a special significance.
But even the known transcription control system, not to mention the whole system of signal transduction and metabolism control, is in principle immense in respect to the mathematical modelling. What will be if this system would be additively complicated for a few of orders? Clearly, such colossal informational redundancy may be biologically useful for system stability, but in what way the system selects a concrete target or agent in each single act of regulation? Answering this question, one must take to account that, in the processes considering here, binding stereospecificity is more or less guaranteed in each version, characteristic times and energies are similar or identical and selection process is realizing in the same compartment.
Using one well known mathematical regularity, we propose the following answer. Competition between some two ligands for certain binding site (receptor) may be pictured in the following form. Let the hypotenuse of a right-angled triangle represents total (maximal) binding energy of a given receptor with a ligand. Than, projection of each leg on the hypotenuse represents a part of binding energy, being realized by the certain ligand in competition with the second certain ligand under given physical and chemical conditions (reagent concentrations, temperature, pressure, pH, ionic force, etc.). Clearly, a) the parts ratio may vary dependently on concrete ligands kind as well as on physical and chemical conditions; b) each ligand may be in competition for a certain target (receptor) with two or more other ligands; c) two or more targets (receptors) may be in competition for each ligand.
Then, let any intracellular regulatory system can be described in the terms of the competitive ligand-receptor interactions (there are not known any reasons because of which this may be not).
The premisses mean that any intracellular regulatory system, in its state space, may be described with a multidimensional system of equations with trigonometric polynomials in their left parts.
Suppose a chain of N agents, where each k-th (k=2, 3, ..., N-1) agent is a ligand for the (k+1)-th agent and, at the same time, a target for the (k-1)-th agent. In a continual approximation, each k-th agent in this chain causes some acceleration (positive or negative) or increment-decrement of energy of the (k+1)-th agent interaction with its target, i.e., with the (k+2)-th agent. Thus, the increment of binding energy of k-th ligand to (k+1)-th target would be a total differential of (k-1) degree of the trigonometric polynomial function describing the corresponding interaction. Taking to account there are numerous multimember feedback loops in the biological regulatory systems, the previous paragraph may be applied to almost all ligand-target interactions.
Note 2. If the chain is initiated by some exogenous influence, then k=1, 2, 3, ..., N, were "1" is the index of the "first", i.e., immediately externally disturbed, agent.
So, one can apply the known regularity of root crystallization of random trigonometric polynomials [8] to different regulatory systems based on the lig-and-receptor interactions. This makes a possible mechanism optimizing the level of informational redundancy in different biological regulatory systems, especially in the transcription control.
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