Ruibo Bio: Detailed description of miRNA characteristics, functions and identification methods

What is miRNA?

MicroRNAs (miRNAs) are single-stranded small-molecule RNAs of about 21-23 bases in length. They are produced by Dicer enzyme processing from a single-stranded RNA precursor of about 70-90 bases in size with a hairpin structure. Unlike siRNA (double strand) but closely related to siRNA. It is speculated that these non-coding small RNAs (miRNAs) are involved in the regulation of gene expression, but the mechanism is different from siRNA-mediated mRNA degradation. The first confirmed miRNA was lin-4 and let-7 , first discovered in nematodes, and several research groups subsequently identified hundreds of miRNAs in a variety of biological species including humans, fruit flies, and plants.

miRNA characteristics

The miRNAs that have been identified are presumably mostly produced by a single-stranded RNA precursor having a hairpin structure and a hairpin structure of about 70 bases, which are processed by Dicer, having a 5' terminal phosphate group and a 3' hydroxyl group. A small RNA fragment of approximately 21-25 nt in size, localized to the 3' or 5' end of the RNA precursor.

Fifteen percent of the 100 new miRNAs identified by the recent three research groups from C. elegans, Drosophila, and Hela cells are highly conserved across the nematode, Drosophila, and mammalian genomes (only 1-2 bases) The difference), colleagues at Lau and Bartel Labs believe that all miRNAs may have orthologs in other species (Ortholog, which refers to those genes that originate from the same ancestor and perform the same function in different organisms). As a category, these similar genes are called "orthologs").

Bantam was first thought to be a genetic locus involved in cell proliferation in Drosophila. It is known that the insertion of several transposons containing an enhancer into a 12.3 kb region spanning this site results in repeated growth of the eye and wing of Drosophila, whereas a transposon-mediated deletion of a 23 kb fragment spanning that site is The mutant flies are smaller than the wild type flies. Cohen and colleagues used a 3.85 kb fragment to introduce a 21 kb fragment-deficient flies into their original size. But the strange thing is that the EST in this 3.85kb fragment has no similar effect. Cohen compared this fragment with the homologous sequence of Anopheles gambiae and found a highly conserved region of 90 bp. The RNA folding program (mfold) found that this conserved sequence can form a hairpin structure, making this segment much like a miRNA. Precursor. This result confirmed that the larvae of the mutant Drosophila lacked a 21 bp bantam miRNA by Northern blot, and the role of bantam miRNA in cell proliferation was confirmed by a series of "functional deletion"-"function recovery" experiments using this 90 bp mRNA precursor. The researchers used a computer program to search for three potential binding sites for bantam in the 3' non-coding region of hid mRNA ( hid is an apoptosis-inducing gene in Drosophila) and confirmed that bantam miRNA inhibits the translation of hid instead of Transcription.

The expression patterns of miRNAs vary. Some worms and Drosophila miRNAs are expressed in all cells at all developmental stages, while other miRNAs are based on a more restrictive phase and temporal expression pattern. There are significant differences in the levels of miRNAs in different tissues and different developmental stages.

miRNA function

Research on microRNAs (miRNAs) is increasing because scientists are beginning to recognize that these ubiquitous small molecules have a broad role in the regulation of eukaryotic gene expression. Most of the hundreds of miRNAs that have been found in species such as nematodes, fruit flies, mice, and humans have the same characteristics as other molecules involved in the regulation of gene expression—the levels of miRNAs are significant in different tissues and stages of development. Difference, this miRNAs expression pattern has differential spatial and temporal expression patterns, suggesting that miRNAs may be important as molecules involved in the regulation of gene expression.

The first confirmed miRNA, lin-4 and let-7 , first discovered in nematodes, can be induced in an unknown way by partial complementation to the 3' non-coding regions (3'UTRs) of the target mRNA target. Protein translation inhibition, which in turn inhibits protein synthesis, regulates the development of nematodes by regulating the translation of a set of key mRNAs (reviewed in Pasquinelli 2002).

The bantam miRNA is the first miRNA to be found to have a proto-oncogene function. In addition to lin-4 and let-7 , some miRNAs are known to be involved in post-transcriptional regulation of genes that play important roles in cell differentiation and tissue development, such as mir-14, mir-23, and the like.

There are two clues in the study of plant miRNAs suggesting that miRNAs may be involved in plant development. First, the expression levels of three miRNAs in the carpel factory ( car ) mutant were significantly decreased. CARPEL FACTORY is a Dicer-like enzyme involved in the development of plants, and its deletion mutants are characterized by defects in embryonic and leaf development. The experimental results suggest that this defect is caused by the lack of processing of miRNAs. The high level of expression of most plant miRNAs in certain tissues also suggests that they may be involved in the development of plant tissues.

Analysis of a subset of miRNAs suggests that miRNAs are involved in a number of important processes in life processes, including early development (Reinhart 2000), cell proliferation, apoptosis, cell death (Brennecke 2003), fat metabolism (Xu 2003), and cell differentiation. (Kawasaki 2003). In addition, one study showed a significant association between a decrease in the levels of two miRNAs and chronic lymphocytic leukemia, suggesting a possible relationship between miRNAs and cancer (Calin 2002).

Due to the extensiveness and diversity of miRNAs, it is suggested that miRNAs may have a wide variety of biological functions. Although the study of miRNAs is still in its infancy, it is speculated that the regulation of gene expression by miRNAs in higher eukaryotes may be as important as transcription factors. One view is that miRNAs may represent a way to regulate gene expression at a newly discovered level.

However, the function of most miRNAs remains a mystery.

How miRNAs work

The first two miRNAs discovered, lin-4 and let-7, are thought to bind to the 3' non-coding region of the target mRNA by incomplete complementation, inducing protein translational inhibition in an unknown way, thereby inhibiting protein synthesis. Block the translation of mRNA. Multiple Drosophila miRNAs have also been found to be partially homologous to the 3' non-coding regions of their target mRNAs. Since the miRNAs and their potential target targets are not completely complementary, it makes it difficult to identify the target site of the miRNA by informatics. Therefore, it is impossible to determine the mode of action of miRNAs, and what mechanism affects the translation of mRNA and how to regulate gene expression. The target and activity mechanism of miRNAs have always been the focus of attention of researchers everywhere.

There is currently one miRNA in the plant that is fully complementary to the three potential target genes (these scarecrow genes encode potential transcription factors), although it is unclear whether these genes are targets of miRNAs, this is the first time that miRNAs have been discovered. Fully complementary to its potential target, it also suggests that miRNAs may contain a similar mode of action as siRNA.

Method for identifying miRNAs

Several research groups have used bioinformatics as a bioinformatics method to conduct research on miRNAs. Because of the presumed degradation of RNA by Dicer, 21-23 bases, 5' hydroxyl and 3' hydroxyl RNA fragments, some laboratories use improved directional cloning methods to screen for the same characteristics. Small molecules - Screening for RNA molecules of a certain size, ligating to 3' and 5' adapters, reverse transcription and amplification by PCR, subcloning and sequencing. The localization and clustering of miRNA precursors on the genome is performed by querying the genome database. This method helps to determine whether miRNAs are degradation products of molecules such as mRNAs, tRNAs, and rRNAs.

Some laboratories use an RNA folding program 'mfold' to determine whether highly conserved regions between C. elegans and C. briggsae contain potential miRNA precursors, and then use Northern Blots to determine whether these miRNAs are actually expressed. It is.

Although hundreds of miRNAs have been identified by biochemical or bioinformatic methods, the identified miRNAs are only a drop in the ocean. Since many of the identified miRNAs have been identified from individual clones, it can be assumed that there are still Many miRNAs are “leaked” during the isolation and identification process, and sequencing is far from enough.

1) Computer RNA assembly method of miRNA

The bioinformatics method searches for new miRNA genes in the genome database based on the fact that mature miRNAs have greater sequence homology in different species and the stem-loop structure of the precursors is quite conserved. The method performs search alignment based on comparative genomics principle and bioinformatics software in the sequenced genome, and then performs RNA secondary structure prediction according to the level of homology, and selects qualified candidate miRNAs and miRNA molecules that have been experimentally identified. A comparative analysis was carried out to determine the distribution and quantity of miRNAs in the species. In recent years, with the continuous development of miRNA prediction methods, the number of miRNAs discovered has increased geometrically. These prediction methods have evolved from simple sequence alignment search to current machine learning algorithms, and programming is becoming more intelligent and complex.

The pri-miRNA transcribed from the human miRNA gene is rapidly processed into a pre-miRNA having a stem-loop structure, which is subsequently cleaved into a miRNA:miRNA* dimer and selectively incorporated into a ribonucleic acid-inducing complex (RISC). And target gene recognition. In similar species, miRNAs are very conserved; but among distant species, miRNAs have some differences, especially in pre-miRNAs. The clarification of the mechanism of action of these miRNAs provides a theoretical basis for the development of predictive software, but it still needs to be constantly repaired and improved. In recent years, several miRNA prediction programs based on these rules have been developed (Table 1.1) and are widely used.

2) miRNA target gene prediction method

The significance of the research of miRNA is undoubted. However, functional studies of miRNAs have been relatively slow compared to the frequent discovery of new miRNAs. To date, of the more than 4,449 miRNAs found, there are only a few dozen miRNAs that define function. A very important reason for the slow progress in miRNA function research is that the target of miRNA action is difficult to determine. In fact, it is very time-consuming to determine the target of miRNA by experimental methods, and there is currently no high-throughput target identification method. Therefore, predicting the target of miRNA by theoretical methods is an ideal way to identify the target of miRNA. The following focuses on the algorithm analysis of several classic prediction software, other software (Table 1.2), please check the corresponding website

Relationship between miRNA and siRNA

The relationship between miRNA and siRNA is confusing. On the surface, one is a non-coding single-stranded small-molecule RNA that is highly conserved in evolution and regulates gene expression through translational inhibition without affecting the stability of the transcript; the other is a double-stranded small-molecule RNA targeting the coding region. Each transcript may have a large number of siRNAs that regulate gene expression after transcription by degrading the target. Since each mRNA template may produce a large number of siRNAs, it is difficult to assign a gene name to each siRNA. miRNAs are highly conserved in the evolutionary process, so giving the same name to an ortholog may help to understand their function, and giving the same name to an unrelated sequence in another species can be confusing.

However, it is speculated that miRNAs are usually cleaved by the Dicer enzyme from a larger (7090 nt) stem-loop structure (hairpin structure) precursor, and Dicer is also responsible for cleaving long double-stranded RNA into siRNA, and both The length is also similar, and there is also regulation of gene expression. Therefore, the relationship between these two types of small RNAs is of particular concern.

Two well-known miRNAs, lin-4 and let-7 , first discovered in nematodes, can be partially induced to bind to the 3' non-coding regions (3'UTRs) of the target mRNA target, inducing protein translation in an unknown way. Inhibition inhibits protein synthesis. This binding does not induce degradation of the mRNA target, that is, as a translational repressor itself does not affect the abundance of the corresponding mRNA, presumably due to incomplete complementarity between the miRNA and the binding site. This is in contrast to siRNA-mediated degradation of mRNA. However, some other miRNAs may mediate the degradation of the target RNA in a manner similar to siRNA. Experiments have shown that introduction of a miRNA that is fully complementary to the let-7 target mRNA target induces degradation of the mRNA target. There are also experimental results indicating that some miRNAs, including Scarecrow miRNAs found in plants, bind to fully complementary mRNA strands to degrade mRNA sequences and inhibit protein synthesis. This suggests that miRNAs can act in the same way as siRNAs, and that the two small RNA RNA pathways may have overlapping parts. This overlap also suggests that siRNAs may also have the same function as miRNAs.

A very interesting experiment confirms this view: Doench and colleagues select a siRNA known to be effective in silencing the CXCR4 gene in vivo, and then insert the corresponding CXCR4 binding site at the 3' end of the luciferase reporter gene - one copy Insert a perfectly matched CXCR4 binding site, another copy inserts 4 only 3' and 5' end matches, and a different CXCR4 binding site in the middle, so that the selected siRNA cannot fully bind to this binding site - - A mismatched ring forming a protrusion in the middle. These two copies were transferred to HeLa cells and gene silencing was induced with siRNA. The results were interesting—both experiments showed a doubling of luciferase activity by more than 10 fold, and RT-PCR and Northern analysis confirmed that the luciferase transcript of the first experiment fell more than 10 times, which is normal. siRNA-mediated RNAi response, degradation of target target mRNA results in decreased expression levels, whereas in the second experiment luciferase transcripts only decreased by a factor of 1.2, this target gene expression level appears to be derived from miRNA-mediated translation Inhibition of decline, rather than siRNA-mediated effects on the stability of mRNA. Experiments show that siRNA may act on mRNA in the form of miRNA. The experimenter also conducted another experiment: changing the base sequence of the mismatched loop in the second experiment does not seem to affect the inhibitory effect, but the higher the degree of matching between the binding sites on the siRNA and the reporter gene, the better the inhibition effect. Increasing the amount of siRNA, the better the inhibition effect - this is the same as the inhibition of siRNA - the only difference is that the perfectly matched binding sites (the mode of action of siRNA) can act independently without affecting each other, but the increase is not complete. The number of paired binding sites (note that 4 CXCR4 binding sites were used in the second experiment) had a significant additive effect on translational inhibition.

Endogenous siRNA has not been found in mammalian cells, and the role of exogenous siRNA-mediated RNAi is a mechanism of resistance. While miRNAs are widely present in mammalian cells, they are theoretically speculated to be involved in multiple regulatory roles. The mechanism of action and the nature of the relationship between these two small things are even more confusing. How to correctly identify siRNA and miRNA in experiments, and even other small RNAs has become a concern.