Analysis of the intermediates formed during splicing of pre-mRNAs in vitro led splicing of exons proceeds sequential to the discovery that via two transesterification reactions. Introns are removed as a lariat-like structure in which the 5 ’G of the intron is joined in an unusual 2,5-phosphodiester bond to an adenosine near the 3 ’ end of the intron. This A residue is called the branch point because it forms an RNA branch in the lariat structure . In each transesterification reaction , one phosphoester bond is exchanged for another . Since the number of phosphoester bonds in the molecule is not changed in either reaction, no energy is consumed. The net result of these two reactions is that two exons are ligated and the intervening intron is released as a branched lariat structure .
Five U-rich small nuclear RNAs (snRNAs), designated U1, U2, U4, U5, and U6 , participate in pre-mRNA splicing . Ranging in length from 107 to 210 nucleotides , these snRNAs are associated with 6 to 10 proteins in small nuclear ribonucleoprotein particles (snRNPs) in the nucleus of eukaryotic cells. Definitive evidence for the role of U1 snRNA in splicing came from experiments which indicated that base pairing between the 5 splice site of a pre-mRNA and the 5 region of U1 snRNA is required for RNA splicing
Involvement of U2 snRNA in splicing initially was suspected when it was found to have an internal sequence that is largely complementary to the consensus sequence flanking the branch point in pre-mRNAs. Compensating mutation experiments, similar to those conducted with U1 snRNA and 5 splice sites , demonstrated that base pairing between U2 snRNA and the branch-point sequence in pre-mRNA also is critical to splicing. the general structures of the U1 and U2 snRNAs and how they base-pair with pre- mRNA during splicing. Significantly, the branch-point A itself , which is not base-paired to U2 snRNA , “bulges out,” allowing its 2 hydroxyl to participate in the first transesterification reaction of RNA splicing
Spliceosomes, Assembled from snRNPs and a Pre-mRNA, Carry Out Splicing According to the current model of pre-mRNA splicing, the five splicing snRNPs are thought to assemble on the premRNA , forming a large ribonucleoprotein complex called a spliceosome. Assembly of a spliceosome begins with the base pairing of the snRNAs of the U1 and U2 snRNPs to the pre-mRNA. Extensive base pairing between the snRNAs in the U4 and U6 snRNPs forms a complex that associates with U5 snRNP . The U4/U6/U5 complex then associates with the previously formed U1/U2/pre- mRNA complex to yield a spliceosome.
After formation of the spliceosome, extensive rearrangements in the pairing of snRNAs and the pre-mRNA lead to release of the U1 and U4 snRNPs . The catalytically active rearranged spliceosome then mediates the first transesterification reaction that forms the 2,5-phosphodiester bond between the 2 hydroxyl on the branch point A and the phosphate at the 5 end of the intron.
Following another rearrangement of the snRNPs, the second transesterification reaction ligates the two exons in a standard 3,5-phosphodiester bond , releasing the intron as a lariat structure associated with the snRNPs. This final intron-snRNP complex rapidly dissociates , and the individual snRNPs released can participate in a new cycle of splicing . The excised intron is then rapidly degraded by a debranching enzyme and other nuclear RNases .
Self-Splicing Group II Introns Under certain non physiological in vitro conditions, pure preparations of some RNA transcripts slowly splice out introns in the absence of any protein . This observation led to recognition that some introns are self-splicing . Two types of self-splicing introns have been discovered: group I introns , present in nuclear rRNA genes of protozoans , and group II introns, present in protein- coding genes and some rRNA and tRNA genes in mitochondria and chloroplasts of plants and fungi . all group II introns fold into a conserved, complex secondary structure containing numerous stem-loops. Self-splicing by a group II intron occurs via two transesterification reactions , involving intermediates and products analogous to those found in nuclear pre- mRNA splicing. The mechanistic similarities between group II intron self-splicing and spliceosomal splicing led to the hypothesis that snRNAs function analogously to the stem-loops in the secondary structure of group II introns .
According to this hypothesis, snRNAs interact with 5 and 3 splice sites of pre mRNAs and with each other to produce a three-dimensional RNA structure functionally analogous to that of group II self-splicing introns.
An extension of this hypothesis is that introns in ancient pre-mRNAs evolved from group II self-splicing introns through the progressive loss of internal RNA structures , which concurrently evolved into trans-acting snRNAs that perform the same functions. Support for this type of evolutionary model comes from experiments with group II intron mutants in which domain V and part of domain I are deleted. RNA transcripts containing such mutant introns are defective in self-splicing , but when RNA molecules equivalent to the deleted regions are added to the in vitro reaction, selfsplicing occurs . This finding demonstrates that these domains in group II introns can be trans- acting, like snRNAs. The similarity in the mechanisms of group II intron self splicing and spliceosomal splicing of pre-mRNAs also suggests that the splicing reaction is catalyzed by the snRNA , not the protein, components of spliceosomes.
Although group II introns can self-splice in vitro at elevated temperatures and Mg 2+ concentrations, under in vivo conditions proteins called maturases , which bind to group II intron RNA, are required for rapid splicing. Maturases are thought to stabilize the precise three-dimensional interactions of the intron RNA required to catalyze the two splicing transesterification reactions . By analogy, snRNP proteins in spliceosomes are thought to stabilize the precise geometry of snRNAs and intron nucleotides required to catalyze pre-mRNA splicing.
Group I Intron
Processing of rRNA and tRNA Approximately 80 percent of the total RNA in rapidly growing mammalian cells (e.g., cultured HeLa cells) is rRNA , and 15 percent is tRNA ; protein-coding mRNA thus constitutes only a small portion of the total RNA. The primary transcripts produced from most rRNA genes and from tRNA genes , like pre-mRNAs , are extensively processed to yield the mature , functional forms of these RNAs. Pre-rRNA Genes Are Similar in All Eukaryotes The 28S and 5.8S rRNAs associated with the large (60S) ribosomal subunit and the 18S rRNA associated with the small (40S) ribosomal subunit in higher eukaryotes are encoded by a single type of pre-rRNA transcription unit . Transcription by RNA polymerase I yields a 45S (13.7-kb) primary transcript (pre-rRNA), which is processed into the mature 28S, 18S, and 5.8S rRNAs found in cytoplasmic ribosomes .
Cloning and sequencing of the DNA encoding pre-rRNA from many species showed that this DNA shares several properties in all eukaryotes. First, the pre-rRNA genes are arranged in long tandem arrays separated by non transcribed spacer regions ranging in length from ≈ 2 kb in frogs to ≈ 30 kb in humans. Second, the genomic regions corresponding to the three finished rRNAs are always arranged in the same 5 ′ → 3 ′ order: 18S, 5.8S, and 28S
Third, in all eukaryotic cells (and even in bacteria), the pre-rRNA gene , as well as the corresponding primary transcript , is considerably longer than the sum of the three finished rRNA molecules . For example, in human cells only about half of the 45S pre-rRNA primary transcript appears in the final rRNA products , whose combined length is about 7.2 kb. The other half, called transcribed spacer RNA , is removed during processing and is rapidly degraded. Discovery of pre-rRNA processing was the first indication that mature cytoplasmic RNAs are derived from larger precursor RNAs synthesized in the nucleus. Both the synthesis and processing of pre-mRNA occurs in the nucleolus. When pre-rRNA genes initially were identified in the nucleolus by in situ hybridization , it was not known whether any other DNA was required to form the nucleolus . Subsequent experiments with transgenic Drosophila strains demonstrated that a single complete pre-rRNA transcription unit induces formation of a small nucleolus .
Thus a single pre-rRNA gene is sufficient to be a nucleolar organizer , and all the other components of the ribosome diffuse to the newly formed pre-rRNA . The structure of the induced nucleolus appears, at least by light microscopy , to be the same as, except smaller than, a normal Drosophila nucleolus containing 200 or so pre-rRNA genes. Small Nucleolar RNAs (snoRNAs) Assist in Processing rRNAs and Assembling Ribosome Subunits Following their synthesis in the nucleolus, nascent pre-rRNA transcripts are immediately bound by proteins, forming pre-ribonucleoprotein particles , or pre- rRNPs. Several ribonucleoprotein particles of different sizes have been extracted from mammalian nucleoli . The largest of these (80S) contains an intact 45S pre-rRNA molecule, which is cut in a series of cleavage and exonucleolytic steps that ultimately yield the mature rRNAs found in ribosomes
During processing, pre-rRNA also is extensively modified, mostly by methylation of the 2 ′ -hydroxyl group of specific riboses and conversion of specific uridine residues to pseudouridine . Some of the proteins in the pre-rRNPs found in nucleoli remain associated with the mature ribosomal subunits , whereas others are restricted to the nucleolus and assist in assembly of the subunits .
The positions of cleavage sites in pre-rRNA and the specific sites of 2 ′ - O - methylation and pseudouridine formation are determined by approximately 150 different small nucleolus-restricted RNA species, called small nucleolar RNAs (snoRNAs), which hybridize transiently to pre-rRNA molecules. Like snRNAs , snoRNAs associate with proteins , forming snoRNPs. One large class of snoRNAs, involved in 2 ′ - O -methylation , contain common sequences bound by the nucleolus-restricted protein fibrillarin . A conserved sequence in these snoRNAs , which is invariably positioned close to methylation sites in the pre-rRNA, is thought to bind a methyltransferase enzyme that modifies the ribose moiety. Another snoRNP, called RNase MRP , catalyzes one of the cleavages by which transcribed spacer sequences are removed from pre-rRNA. The associated snoRNA is homologous to the RNA of RNase P involved in tRNA processing. Based on this homology, the cleavage reaction is thought to be catalyzed by the MRP snoRNA. There is strong evidence that RNase P performs one of the pre-rRNA cleavages as well.
Some snoRNAs are expressed from their own promoters by RNA polymerase II or III . Remarkably, however, the large majority of snoRNAs are spliced-out introns of genes encoding functional mRNAs . Unlike pre-rRNA genes , 5S-rRNA genes are transcribed by RNA polymerase III in the nucleoplasm outside of the nucleolus . Without further processing, 5S RNA diffuses to the nucleolus , where it assembles with the 28S and 5.8S rRNAs and proteins into large ribosomal subunits . When assembly of ribosomal subunits in the nucleolus is complete, they are transported through nuclear pore complexes to the cytoplasm , where they appear first as free subunits .
The DNA in the protozoan Tetrahymena thermophila contains an intervening intron in the region that encodes the large pre-rRNA molecule . Careful searches failed to uncover even one pre-rRNA gene without the extra sequence , indicating that splicing is required to produce mature rRNA in these organisms. Subsequent studies showing that the pre-rRNA was spliced at the correct sites when incubated by itself, without assistance from any protein , provided the first indication that RNA can function as a catalyst , like enzymes . Following the discovery of self-splicing in Tetrahymena pre-rRNA , a whole raft of self-splicing sequences were found in pre-rRNAs from other single-celled organisms, in mitochondrial and chloroplast pre-rRNAs. The self-splicing sequences in all these precursors, referred to as group I introns , use guanosine as a cofactor and can fold by internal base pairing to juxtapose closely the two exons that must be joined . Clearly, in the self-splicing introns , RNA functions as a ribozyme , an RNA sequence with catalytic ability .
The group I intron within the pre-rRNA of Tetrahymena and certain other organisms is unrelated to the transcribed spacer sequences that separate the 18S, 5.8S, and 28S regions in the majority of organisms. In particular, the self-splicing mechanism that removes group I introns differs from the cleavage mechanism by which spacer sequences are removed during processing of pre-rRNA Mutational and biochemical experiments are under way to determine which residues are critical in catalyzing the two transesterification reactions leading to splicing.
All Pre-tRNAs Undergo Cleavage and Base Modification Mature cytosolic tRNAs , which average 75 – 80 nucleotides in length, are produced from larger precursors (pre-tRNAs) synthesized by RNA polymerase III in the nucleoplasm . Mature tRNAs also contain numerous modified bases that are not present in tRNA primary transcripts . Cleavage and base modification occur during processing of all pre-tRNAs . Some pre-tRNAs contain one or more introns that are spliced out during processing. A 5 ′ sequence of variable length that is absent from mature tRNAs is present in all pre-tRNAs .
These extra 5 ′ nucleotides are removed by the ribonuclease P (RNase P ), a ribonucleoprotein endonuclease . The RNase P polypeptide increases the rate of cleavage by M1 RNA, allowing it to proceed at physiological Mg2+ concentrations. A 14-nucleotide intron (blue) in the anticodon loop is removed by splicing. A 16- nucleotide sequence (green) at the 5 ′ end is cleaved by RNase P . U residues at the 3 ′ end are replaced by the CCA sequence (red) found in all mature tRNAs . Numerous bases in the stem-loops are converted to characteristic modified bases (yellow). Not all pre-tRNAs contain introns that are spliced out during processing, but they all undergo the other types of changes shown here. D = dihydrouridine ; Ψ = pseudouridine .0
About 10 percent of the bases in pre-tRNAs are modified enzymatically during processing. Three types of base modifications occur : 1) replacement of U residues at the 3 ′ end of pre-tRNA with a CCA sequence, which is found at the 3 ′ end of all tRNAs 2) addition of methyl and isopentenyl groups to the heterocyclic ring of purine bases and methylation of the 2 ′ -OH group in the ribose of any residue; and 3) conversion of specific uridines to dihydrouridine , pseudouridine , or ribothymidine residues. Processing of pre-tRNA, like mRNA processing, occurs in the nucleoplasm . The mature tRNAs then are transported to the cytoplasm through nuclear pore complexes. Interestingly, U6 snRNA, another RNA synthesized by RNA polymerase III, is a component of the spliceosome and remains in the nucleus, whereas tRNAs are efficiently transported to the cytoplasm . Most likely, mature tRNAs in the nucleus, like mature mRNAs and rRNAs, are bound by specific proteins that facilitate their transport through nuclear pores . Once in the cytoplasm, tRNAs are passed between aminoacyl-tRNA synthetases , elongation factors, and ribosomes during protein synthesis. Thus tRNAs generally are associated with proteins and spend little time free in the cell, as is also the case for mRNAs and rRNAs.
Splicing of Pre-tRNAs Differs from Other Splicing Mechanisms Comparison of the sequences of tRNA genes with the sequences of the corresponding cytosolic tRNAs has shown that some eukaryotic nuclear tRNA genes contain introns . For example, the pre-tRNA expressed from the yeast Tyr ) gene contains a 14-base intron that is not present in tyrosine tRNA (tRNA mature tRNA Tyr Some archaeal tRNA genes also contain introns. The introns in nuclear pre- tRNAs are shorter than those in pre-mRNAs , and they do not contain the splice- site consensus sequences found in pre-mRNAs. Pre-tRNA introns also are clearly distinct from the much longer self-splicing group I and group II introns found in chloroplast and mitochondrial pre- rRNAs . The mechanism of pre-tRNA splicing, outlined in Figure 11-53, differs in several ways from the mechanisms utilized by self-splicing introns and spliceosomes.
For instance, during pre-tRNA splicing the intron is excised in one step rather than two; GTP and ATP are required; a 2 ′ , 3 ′ -cyclic monophosphate forms on the cleaved end of the 5 ′ exon ; and the process is catalyzed by proteins (enzymes) rather than RNA . Certain mutations in pre-tRNA that change its secondary structure prevent the splicing reaction , indicating that pre-tRNA molecules must be folded into a particular secondary structure for intron excision to occur. Since introns always are found in the anticodon loop of pre-tRNAs , pre-tRNAs most likely are folded similarly to mature tRNAs , thereby bringing the two intron-exon junctions into proximity . When intron-containing yeast tRNA genes are microinjected into Xenopus oocyte nuclei, correctly processed tRNAs are produced. This finding indicates that enzymatic systems for cleaving, modifying , and splicing pre-tRNAs have been conserved over a wide evolutionary range.
First, the pre-tRNA is cleaved at two places, on each side of the intron, thereby excising the intron. The cleavage mechanism generates a 2 ′ ,3 ′ -cyclic phosphomonoester at the 3 ′ end of the 5 ′ exon. The multistep reaction joining the two exons requires two nucleoside triphosphates: a GTP, which contributes the phosphate group (yellow) for the 3 ′ → 5 ′ linkage in the finished tRNA molecule ; and an ATP, which forms an activated ligase-AMP intermediate . The 2 ′ - phosphate on the 5 ′ exon is removed in the final step
RNA EDITING RNA editing can be broadly defined as any site-specific alteration in an RNA sequence that could have been copied from the template , excluding changes due to processes such as RNA splicing and polyadenylation . Changes in gene expression attributed to editing have been described in organisms from unicellular protozoa to man , and can affect the mRNAs, tRNAs, and rRNAs present in all cellular compartments. These sequence revisions , which include both the insertion and deletion of nucleotides , and the conversion of one base to another , involve a wide range of largely unrelated mechanisms .
ADARs Editing of adenosine (A) to inosine (I) in double-stranded RNA , catalyzed by adenosine deaminases acting on RNA ( ADARs ), is one dynamic modification that in a combinatorial manner can give rise to a very diverse transcriptome . Since the cell interprets inosine as guanosine (G), editing can result in non- synonymous codon changes in transcripts as well as yield alternative splicing , but also affect targeting and disrupt maturation of microRNA . ADAR editing is essential for survival in mammals but its dysregulation can lead to cancer . ADAR1 is for instance over expressed in, e.g., lung cancer , liver cancer , esophageal cancer and chronic myoelogenous leukemia , which with few exceptions promotes cancer progression . In contrast, ADAR2 is lowly expressed in e.g. glioblastoma , where the lower levels of ADAR2 editing leads to malignant phenotypes . Altogether, RNA editing by the ADAR enzymes is a powerful regulatory mechanism during tumorigenesis .
MECHANISMS OF INSERTION/DELETION EDITING The internal insertion of nucleotides has been observed in mitochondrial RNAs from kinetoplastid and amoebid protozoa , myxomycetes, chytriomycete fungi, and nematodes. Indeed, insertional editing can occur either post-transcriptionally or cotranscriptionally . 1. Posttranscriptional Nucleotide Insertion/Deletion The term RNA editing was first coined by Benne and colleagues to describe the insertion of uridines into the cytochrome oxidase subunit II mRNA in kinetoplasts of Trypanosoma brucei and Crithidia fasciculata . The global nature of such frame shifting events within the kinetoplastid protozoa was soon established by the identification of additional examples in these species and in Leishmania tarentolae and later extended to more distant relatives.
Deletion of uridine residues was also observed, albeit at lower frequency, within each of these species. The extent of editing was shown to vary from the insertion of a few nucleotides to extensive insertion/deletion of uridine residues ( pan-editing ) in which over 50% of the final mRNA product is the result of RNA editing . The notion of “ editing” sequence at the RNA level was originally met with skepticism , based largely on the absence of any obvious template that could be Used to direct uridine insertion/deletion . The identification of guide RNAs (gRNAs) as the likely source of the missing information quickly led to the formulation of two general models by which accurate insertion and deletion of nucleotides could be achieved . These models, cleavage-ligation and transesterification , made different predictions as to the expected reaction intermediates and stimulated a flurry of Experimentation and debate. Development of in vitro systems capable of carrying out uridine deletion (177, 178) and insertion (36, 49, 94) permitted direct testing of particular features of individual models, resulting in the eventual acceptance of the cleavage-ligation pathway as the mechanism of kRNA editing (4, 73, 195).
Nuclear export of mRNA Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression . In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein ( mRNP ) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation . The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery , thereby ensuring accuracy and swiftness of gene expression .
The molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. Nuclear pore complexes ( NPCs ), which perforate the NE , are the main gateways through which RNAs and protein s are delivered to their proper destinations. The NPC is composed of approximately 30 distinct proteins that are collectively known as nucleoporins. A subset of nucleoporins that line the central transport channel contains phenylalanine-glycine (FG)-repeat sequences , which emanate to the inside of the channel and form a dense hydrophobic meshwork that functions as a barrier limiting the improper exchange of soluble macromolecules between the nucleus and the cytoplasm . Thus, nucleo-cytoplasmic transport of RNAs and proteins requires specific transport receptors to break this barrier. The importin/karyopherin- β family of proteins comprise the prototypical transport receptor family that mediates nucleo-cytoplasmic movement of most proteins and small non-coding RNAs , such as tRNA , uridine-rich small nuclear RNA ( UsnRNA ), and miRNA .
Nuclear export of mRNAs is a unique process that does not directly rely on the functions of the importin/karyopherin- β transport receptor family and Ran . Instead, it requires the evolutionarily conserved heterodimeric transport receptors Tap-p15 (also called Nxf1-Nxt1 ) in metazoans and Mex67-Mtr2 in yeast. Both Tap-p15 and Mex67-Mtr2 are RNA binding proteins , but they bind nonspecifically to RNA in vitro and are not able to distinguish different RNAs on their own. To circumvent this problem, a series of mRNA-binding proteins participate in this process. The conserved transcription-export (TREX) complex, which consists of the THO subcomplex and Uap56 and Aly/REF plays an important role in selection of mRNAs by Tap-p15 and Mex67-Mtr2 . The RNA-binding components of the TREX complex , including yeast Yra1 and mammalian Aly/REF , directly interact with the export receptor heterodimers , thereby functioning as adaptors.
In addition, in yeast, the serine-arginine rich (SR) proteins Npl3, Gbp2 and Hrb1are associated with the TREX complex and the mRNA binding protein Nab2 also interact with Mex67-Mtr2 and probably function as adaptors . In mammalian cells , the SR proteins 9G8 and SRp20, as well as numerous mRNA-binding proteins, have been proposed to play a similar role. Recruitment of adaptor proteins to mRNPs is coupled with transcription and processing, causing mRNPs to be licensed to the mRNA-specific export pathway upon the completion of nuclear processing . Thus, transcription by RNA polymerase II (RNAPII) is a key determinant allocating mRNA to the appropriate export pathway.
protein factors capping, splicing During transcription, required for and cleavage/polyadenylation are recruited to the nascent transcript , forming an mRNP . The 5' end of the mRNA is capped early in this process via an interaction between the capping enzyme and RNA polymerase II (RNAPII). Factors involved in splicing and cleavage/polyadenylation are also co- transcriptionally loaded onto the pre-mRNA. The TREX complex and a subset of the SR proteins, which are engaged in nuclear export, are recruited to the nascent mRNA via interactions with the transcription and processing factors . The nuclear export receptor Tap-p15 (Mex67-Mtr2 in yeast) in turn gains access to the mRNA via interactions with these factors as adaptors. The nuclear export receptor heterodimer facilitates the translocation of mRNPs through its interaction with FG-repeat containing nucleoporins.
During the process of the nuclear mRNA biogenesis, the structure and the composition of the mRNP change drastically and these alterations in the physicochemical properties also help the mRNP translocate through the NPC. The mRNA export factors are then dissociated from the mRNP by factors associated with the NPC to prevent the return of the mRNP to the nucleus . The exported mRNA then directs protein translation in the cytoplasm.
Regulatory RNA And Non Coding RNA (nc RNA) Though 80% of the human genome is transcribed into RNA , majority of RNA lacks protein coding potential and referred as “non -coding RNA” ( ncRNA ). The mammalian transcriptome is much more complex and their transcription is regulated by developmental stages . The continuing discovery of new classes of regulatory ncRNAs suggests that RNA has continued to evolve along with proteins and DNA . The ncRNAs are divided into two major groups based on an arbitrary threshold of 200 nucleotides (nt) namely 1) short ncRNAs (sncRNA) and 2) long ncRNAs (lncRNAs) . The sncRNAs include functional RNAs such as t-RNAs, r-RNAs and snRNAs which are involved in transcriptional and translational regulation. The short ncRNAs also include different regulatory RNAs such as microRNAs ( miRNAs ), small interfering RNAs (siRNAs) and P-element-induced wimpy testis (PIWI) interacting RNAs (piRNAs), all of which regulate gene expression.
miRNAs The miRNAs are 20 – 30 nucleotides long and generated from sense and antisense DNA strands. MicroRNAs are found in the plant and animal branches of Eukaryota and are encoded by a bewildering array of genes . They induce mRNA degradation or translational repression , which in turn result in the alteration of gene expression . About 60% of translated protein coding genes are negatively regulated by miRNAs . Some transcripts are regulated by a single miRNA , while others are regulated by more than one miRNAs . In addition to the transcriptional gene regulation , miRNAs play important roles in pivotal biological processes such as cell proliferation, cell differentiation, development, and cell death.
The miRNA biogenesis and mechanism of action The process of miRNA biogenesis is quite characteristic for the ncRNAs subclass . Based on cellular requirement, the primary miRNA transcript (pri-miRNA) is first transcribed from the DNA by RNA polymerase II and transcripts are capped and polyadenylated. They are characterized by one or many stem-loop hairpins which encompass the functional mature miRNA in their stem . In animals, the first step occurs in nucleus , in which the pri-miRNA upon recognition by two nuclear enzymes , Drosha and DGCR8 is processed into dsRNA molecule containing one or more hairpins of approximately 70 nucleotides long , which are called as precursor miRNAs (pre-miRNAs). Then they are exported to the cytoplasm by the nuclear export protein exportin-5 . In cytoplasm, the pre-miRNA is recognized and processed by the RNase III enzyme, Dicer which removes the hairpin loop resulting in 20 – 23 nt dsRNA (miRNA-miRNA*) molecule. When the complementarity between the miRNA bound to Argonaute protein 1 (Ago1) protein and the target m-RNA is high, miRNA tailing and 3 ′– 5 ′ trimming occurs .
The RNA induced silencing ( RISC) complex then targets the mRNA transcript based on sequence complementarity between the miRNA sequence and nucleotides in the 3 ′ untranslated regions (3 ′ UTR) of the target mRNAs. The binding of the the RNA-induced silencing complex (RISC) to its target leads to direct Ago- mediated cleavage of the target and causes mRNA degradation if the homology between miRNA and its target mRNA is extensive. Initially, it has been showed that miRNAs mainly target the 3 ′ UTRs of mRNAs , but recently, it was found that miRNA target sites also been located in the 5 ′ UTRs and even in coding regions of some of the target mRNAs . For example, mir-148 targets on the coding regions of DNMT3B .
Non repressed mRNAs recruit initiation factors and ribosomal subunits and form circularized structures that enhance translation (top). When miRISCs bind to mRNAs, they can repress initiation at the cap recognition stage (upper left) or the 60S recruitment stage (lower left). Alternatively, they can induce deadenylation of the mRNA and thereby inhibit circularization of the mRNA (bottom). They can also repress a postinitiation stage of translation by inducing ribosomes to drop off prematurely (lower right). Finally, they can promote mRNA degradation by inducing deadenylation followed by decapping .
siRNA The canonical inducer of RNAi is long, linear, perfectly base paired dsRNA, introduced directly into the cytoplasm or taken up from the environment. These dsRNAs are processed by Dicer into the siRNAs that direct silencing. The siRNAs were originally observed during trans gene and virus-induced silencing in plants, consistent with a natural role in genome defense. In 2002 and 2003, centromeres, transposons, and other repetitive sequences were uncovered as another wellspring of siRNAs.
siRNA Biogenesis and Mechanism In cytoplasm, the small RNA duplex molecules produced by the action of Dicer , creates a RNA duplexes with 2-nt overhangs at their 3 ′ ends and phosphate groups at their 5 ′ ends . Only one of the two strands of dsRNA acts as a guide strand and directs gene- silencing while, the other strand incorporates into the RNA-induced silencing complex (RISC) containing the Argonaute proteins (Ago2) and the GW182. The siRNAs are recognized by Argonaute protein 2 ( Ago2 ). The pathways have been characterized in Drosophila and in humans . The siRISC assembly in Drosophila is nucleated by the R2D2/Dicer-2 heterodimer , which binds an siRNA duplex and then progresses by the addition of unknown factors to form the RISC-loading complex (RLC) . The RLC then assembles into pre-RISC , with the siRNA still in duplex form. The pre-RISC formation is the first step that requires Ago2 , the Drosophila Ago protein. The Ago2 then cleaves the passenger strand , leading to its ejection and the conversion of the entire assembly into the 80S holo-RISC .
RISC assembly in humans has also been characterized biochemically and appears to be a simpler process. Three proteins — Dicer, TRBP, and Ago-2 — associate with each other even in the absence of the dsRNA trigger . This trimer, also referred to as the RISC-loading complex , is capable of binding dsRNA , dicing it into an siRNA, loading the siRNA into Ago-2, and discarding the passenger strand to generate functional RISC . Additional proteins associate with Ago complexes from human cells, but they do not appear to be essential for RISC loading or target cleavage . In many species, the siRNA populations that engage a target can be amplified by the action of RNA-dependent RNA polymerase (RdRP) enzymes, strengthening and perpetuating the silencing response .
Different categories of siRNAs can depend upon different proteins for their function, indicating that they rely on different biogenesis and RISC assembly pathways. This is particularly true in plants, where viral siRNAs, transgene siRNAs, and tasiRNAs have highly distinct cofactor requirements. During the canonical RNAi pathway , the siRNA guide strand directs RISC to perfectly complementary RNA targets , which are then degraded. The RNA degradation is induced by the PIWI domain of the Ago protein. This ‘ ‘slicer’’ activity is very precise : the phosphodiester linkage between the target nucleotides that are base paired to siRNA residues 10 and 11 is cleaved to generate products with 5 ’ -monophosphate and 3 ’ -hydroxyl termini. Once the initial cut is made, cellular exonucleases attack the fragments to complete the degradative process . The newly generated 3 ’ end of RISC cleavage products is also a substrate for oligouridylation , which can promote exonucleolytic targeting . The target dissociates from the siRNA after cleavage, freeing RISC to cleave additional targets. In some cases, highly purified forms of RISC fail to cleave their targets with multiple turnover, suggesting that extrinsic factors promote product release, which is likely to be driven by ATP hydrolysis .
RNA i RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids , and regulates the expression of protein-coding genes . This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics , therapeutic intervention , agriculture and other areas . Investigations on diverse organisms, labeled variously as PTGS in plants , RNAi in animals, quelling in fungi , and virus-induced gene silencing , have converged on a universal paradigm of gene regulation. The critical common components of the paradigm are that (i) the inducer is the dsRNA (ii) the target RNA is degraded in a homology dependent fashion (iii) the degradative machinery requires a set of proteins which are similar in structure and function across most organisms.
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