Small Regulatory RNAs

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Contents

Introduction

The discoveries in last 5 years made it clear that cell regulatory network is much larger and more complex than it seemed earlier. It was discovered that there is broad class of small regulatory RNAs that play important role in many (if not all) processes in cell.

Classification of Small Regulatory RNAs

  • siRNA - short interfering RNA [1]
  • miRNA - micro RNA [1]
  • snoRNA - small nucleolar RNA [1]
  • smRNA - small modulatory RNA [1]
  • tncRNA - tiny noncoding RNA

RNA and embryogenesis

MicroRNA

The discovery of Micro RNA

MicroRNA was discovered in early 1981 by Martin Chalfie et al. They observed that mutations in lin-4 gene (since it affect cell lineage) of C.Elegans led to continued synthesis of larval-specific cuticles. [2]

In 1989 it was shown by Victor Ambros that lin-4 inhibits lin-14 and lin-28 genes, so that it triggers the activation of lin-29 and it switches the development of larva to adult. [2]

In 1991 it was found that mutations (deletion) in 3'UTR region of lin-14 gene leads to abnormal protein accumulation in larval. So, it was shown that this region is regulatory element of protein translation. [2]

In 1993 Lee at. al and Wightman et.al have shown that lin-4 doesn't encode for any protein and they also found two RNAs 22 and 61 nt long, that are transcribed by this gene. They also found out that these RNAs contain nucleotide sequence, complementary of that in 3'UTR region of lin-14.[2].

In 2000 Reinhart et.a.have found that another gene - let-7 also codes 21-nt RNA and this RNA has complementary sequence to the 3'UTR region of lin-14, lin-28, lin-41, lin-42 and daf-12 genes. So, they proposed that let-7 regulates activity of these genes by RNA-RNA interaction. [2]

In 2001 several research groups (Lagos-Quintana et al., Lau et al., Lee and Ambros) have found many other small RNA (in different organisms) that play similar potentially regulatory role by RNA-RNA interaction.[2]

What is MicroRNA

MicroRNA is small 17-24 nucleotides long RNA, that participates in posttranscriptional regulation. miRNAs can regulate more than 30% all of human genes. The sequences of many of the miRNAs are evolutionary concerved and highly homologous among organisms.

Genomics of MicroRNA

There are 2 ways of presenting of microRNA in the genome. THe first one is when microRNAs are located in the introns of protein-coding mRNA (40%) or within the noncoding RNAs (10%). The second one is when microRNAs are presented as a single transcriptional unit with specific promotor (50%). MiRNAs can exist in the genome as individual or clustered units.

Pathway of MicroRNA Processing

At the first step primary MicroRNA is transcribed from genome, using RNA Polymerase II - the same way as any mRNA is transcribed. After that pri-miRNA contains cap on 3' region and polyadenylated (has sequense of adenyl nucleotides) at 5' end. At this time it doesn't differ from other mRNA. Pri-miRNA forms one or more hairpin (stem and loop) structures and can be hundreds or thousands nt long. [2]

At the second step (cropping), pri-miRNA is processed in nucleus by a special protein complex containing Drosha. Drosha is a nuclear Ribonuclease III endonuclease. This protein complex cleaves pri-miRNA at the stem of each stem-loop part to one or more hairpin-like molecules. These resulting molecules are called precursor miRNA or pre-miRNA. Each pre-miRNA consists of double-stranded stem and loop and is 70-100 nt long. The 3' hydroxyl terminy of the stem overhangs 5' end by 2 or 3 nucleotides.[2]

Drosha is the part of larger complex that is called "Microprocessor". This complex is a heterotetramer, consisting of two Drosha and two DGCR8 (Di George syndrome critical region gene 8) molecules in human. In Drosophila there are Pasha molecules instead of DGCR8 in this complex.[2]

At the third step pre-miRNA is exported by Exportin 5 from nucleus to cytoplasma. Exportin 5 is heterodimer Exp5/RanGTP and it makes hetertrimer with pre-miRNA. After this complex is exported through the nuclear pore to the cytoplasm, RanGTP is hydrolized to RanGDP and pre-miRNA is released from the complex. [2]

At the fourth step pre-miRNA is processed by Dicer. Dicer is cytoplasmic RNase III endonuclease. Dicer cleaves the loop from pre-miRNA molecule. As the result there is double-stranded RNA (miR duplex) and each strand has 2 nt overhang at their 3' ends. The length of each strand is 18-22 nt. It seems possible that miR duplex is also unwound by the Helicase domain of Dicer, so that only one end of the duplex remains base-paired. The mature RNA will be presented by that half of the duplex which has relatively unstable base-pairs at the 5' end.

At the fifth step miRNA is processed by the RNA-induced silencing complex (RISC). RISC contains Ago1 or Ago2??? protein During this processing one of 2 strands of miRNA is disconnected from it, so that only one strand remains in this RNA-protein complex. So this complex is ready now to find target RNAs ans silence them.

How MicroRNA silences target RNAs

RISC/miRNA complex can silence target RNAs by 2 ways.

The first way is cleavage of the target mRNA. It happens usually in the case of perfect complementarity of miRNA and target mRNA. In this case the PIWI domain of Ago protein that contains the 5' end of miRNA, connects to the target RNA, that contains antisence sequence of nucleotides. After that the zipper made by the 2 RNAs "closes" at the area of PAZ domain. After that the target RNA is cleaved at the position about ??? nucleotides from miRNA 5' end.

The perfect pairing among nucleotides 1-8 is required for stable miRNA to target mRNA interaction.

The second way is repressing of translation of mRNA. In this case miRNA connects to mRNA at untranslated region (UTR) of target mRNA. After that it somehow interacts with ribosome so that translation is not initiated. The exact mechanism of this interaction remains unclear yet. It was also shown that after connecting RISC-miRNA complex to target mRNA the resulting complex is relocalised to P-bodies. P-bodies (processing bodies) are known as cytoplasmic aggregates that store, decap and degrade mRNAs. Localizing mRNA within P-body can also inhibit its translation.

Methods for finding of miRNA

  • Computational method. By analyzing genome sequence and finding stem-loop structures in it. The number of miRNA genes is estimated as 1% of metazoan genome. [2]
  • In situ hybridization using locked-nucleic acid-modified oligonucleotide probes have been utilized to determine the temporal and spatial expression pattern of miRNAs. [6]
  •  ???Northern blotting and a more sensitive PCR method (Shmitgen at al. 2004) [2]

How many miRNAs are there?

Analyis of human genome shows that there can be about 1000 different miRNAs. 587 of them are already identified. See: [1]

MiRNA databases

  • Sanger Insitute MicroRNA database and registry - it contains information about all known microRNAs for many different species and information about target genes. It is updated on regular basis. As on October 2006 it contained information about 4361 miRNA from 40 different species from viruses to mammals. It also helps to denote the name to newly discovered miRNAs.
  • RNAdb - This database is a comprehensive mammalian noncoding RNA database containing sequences and annotations for tens of thousands of noncoding RNAs

Role of microRNA in Development, Differentiation and Organogenesis

MicroRNA in embryo development

MiRNAs play important role in normal embryonic development. The essential role of miRNAs is to inhibit the expression of genes that are not required for particular developmental stages or which may promote alternative differentiation pathways. There were also experiments showing the importance of miRNA in development by studying of Dicer-null ES cells. Such cells doesn't process miRNA and result in early lethality of mutant embryos and in-vitro they didn't differentiate, but only formed cell aggregates. It was indicated that Dicer mutated embryos show lethality by early development arrest. This also lead to abnormalities in differentiation and morphogenic processes. Analysis of MiRNAs expressing profile reveals that there is unique set of MiRNAs expressed in each stage of embryo development.

MicroRNA and Embryonic Stem Cells differentiation

It was found in 2003 that there are at least 6 miRNAs in mice (miR-290, miR-291-as, miR-292-as, miR-293, miR-294, miR-295) that are expressed in undifferentiated embryonic stem (ES) cells, but are not expressed after ES cells are cultured for 14 days and are differentiated. Similar miRNAs were found in human ES cells. [4] Hence, a set of stem cell-specific miRNAs is established. At the same time this set is specific only for undifferentiated ES cells but not for postimplantation cell type. Comparing miRNAs signatures in somatic cells it was shown that miRNAs arrays are unique as well. It is necessary to regulate coordinated translation of mRNAs and transcription of miRNAs during the complex process of multicellular organism development. Thus the linkage between these two processes seems to be essential for the correct developmental specification.[5]

Role of DICER and miRNAs in tissue morphogenesis

It is suggested that miRNAs activity is associated with regions of active morphogenesis. The set of tissue-specific miRNAs was experimentally determined (for example, miR-200 and miR-19/20 families are specific to epidermis, miR-199 family - for hair follicles).[5] (miR-1, miR-133, miR-206 are enriched in skeletal and heart muscles of mice and humans. miR-1 enhances myogenesis by targeting HDAC4, which has been shown to inhibit muscle differentiation, while miR-133 targets SRF, which promotes muscle differentiation). [6] It can be suggested that miRNAs can modulate the expression of factors that control cell proliferation and differentiation. The balance between proliferation and differentiation is essential for proper organogenesis.

MiRNAs in apoptosis

The study of Dicer mutated cells reveals the importance of miRNAs in apoptosis mechanism. miRNAs trigger the apoptotic pathway in Drosophila by inducing the expression of the cell death activators Ripper, Hid and Grim and the caspase Dronc. Experiments with mammalian cells have identified that miRNAs can play role in apoptosis and/or proliferation of these cells. MiRNAs can induce or inhibit both of these processes. For example, in the case of chronic lymphocytic leukemia and colon cancer miR-15a and miR-16-1 have been shown to target the antiapoptotic gene transcripts BCL2 in B cells, and RARS in pituitary adenoma cells. Thus, it appears that miRNAs promote apoptosis in normal tissue. MiRNAs promote cell survival and proliferation through inhibiting apoptosis.[5]

MiRNAs in cancer

About the half of miRNAs genes and gene clusters are located near fragile sites (FRAs) and cancer-associated regions (CAGRs) - the sites where translocations, deletions, amplifications, DNA damages often occur. As a consequence all these events affect miRNAs genes. Deletion or down-regulation of miRNAs gene containing DNA lead to inhibition of apoptosis and promote the cancer. MiRNAs were identified to act as tumor suppressor genes. It was observed the malfunction of miRNA precursor processing (reduced DICER expression) in cancer cells. The overexpression of some miRNAs genes in combination with reduced expression level of other miRNAs can lead to cancer as well.[5]

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