RNA interference (RNAi) was discovered unexpectedly during attempts to experimentally manipulate the expression of specific genes. Researchers tried to inhibit the expression of a gene in C. elegans by microinjecting a single-stranded, complementary RNA that would hybridize to the encoded mRNA and prevent its translation, a method called antisense inhibition. But in control experiments, a perfectly base-paired double-stranded RNA a few hundred base pairs long was much more effective at inhibiting expression of the gene than the antisense strand alone. Similar inhibition of gene expression by introduced double-stranded RNA was soon observed in plants. In each case, the double-stranded RNA induced degradation of all cellular RNAs containing a sequence that was exactly the same as that of one strand of the double-stranded RNA. Because of the specificity of this technique in targeting mRNAs for destruction, it has become a powerful experimental tool for studying gene function.

Subsequent biochemical studies with extracts of Drosophila embryos showed that a long double-stranded RNA that mediates RNA interference is initially processed into a double-stranded short interfering RNA (siRNA). The strands in siRNAs contain 21–23 nucleotides hybridized to each other so that the two bases at the 3′ end of each strand are unpaired. Further studies revealed that the cytoplasmic double-stranded RNA–specific ribonuclease that cleaves long double-stranded RNA into siRNAs is the same Dicer enzyme involved in processing pre-miRNAs after their export to the cytoplasm (see Figure 1). This discovery led to the realization that RNA interference and miRNA mediated repression of translation and target-mRNA degradation are related processes. In both cases, the mature short single-stranded RNAs, either siRNAs or miRNAs, are assembled into RISC complexes in which they are bound by an Argonaute protein. What distinguishes a RISC complex containing an siRNA from one containing an miRNA is that the siRNA base-pairs perfectly with its target RNA and in duces its cleavage, whereas a RISC complex associated with an miRNA recognizes its target through imperfect base pairing and results in inhibition of translation and a slower form of target-mRNA degradation (see Figure 2).

Fig1. Processing of miRNA. This diagram shows transcription and processing of the miR-1-1 miRNA. The primary miRNA transcript (pri-miRNA) is transcribed by RNA polymerase II. The nuclear double-stranded RNA–specific endoribonuclease Drosha, with its partner, double-stranded RNA–binding protein DGCR8 (Pasha in Drosophila), makes the initial cleavages in the pri-miRNA, generating a ~70-nucleotide pre-miRNA that is exported to the cytoplasm by nuclear transporter exportin 5. The pre-miRNA is further processed in the cytoplasm by Dicer, in conjunction with the double-stranded RNA–binding protein TRBP (Loquacious in Drosophila), into a double stranded miRNA with two-base single-stranded 3′ ends. Finally, one of the two strands is incorporated into a RISC complex, where it is bound by an Argonaute protein. See P. D. Zamore and B. Haley, 2005, Science 309:1519.

Fig2. Base pairing with target RNAs distinguishes miRNA and siRNA. (a) miRNAs hybridize imperfectly with their tar get mRNAs, repressing translation of the mRNA. Nucleotides 2–7 of an miRNA (highlighted blue) are the most critical for targeting it to a specific mRNA. The CXCR4 miRNA shown at the bottom is a synthetic oligonucleotide introduced into cells by transfection. (b) siRNA hybridizes perfectly with its target mRNA, causing cleavage of the mRNA at the position indicated by the red arrow, triggering its rapid degradation. See P. D. Zamore and B. Haley, 2005, Science 309:1519.

AGO2 is the protein responsible for the cleavage of target RNA. One domain of the protein is homologous to the RNase H enzymes that degrade the RNA of an RNA-DNA hybrid. When the 5′ end of the siRNA of a RISC complex base-pairs precisely with a target mRNA over a distance of one turn of an RNA helix (10–12 base pairs), this domain of AGO2 cleaves the phosphodiester bond of the target RNA across from nucleotides 10 and 11 of the siRNA (see Figure 2b). The cleaved RNAs are released and subsequently degraded by cytoplasmic exosomes and the XRN1 5′→3′ exoribonuclease. If base pairing is not perfect, the AGO2 domain does not cleave or release the target mRNA. Instead, if several miRNA-RISC complexes associate with a target mRNA, its translation is inhibited, and the mRNA becomes associated with P bodies, where, as mentioned earlier, it is degraded by a different and slower mechanism than the degradation pathway initiated by RISC cleavage of a perfectly complementary target RNA.

When double-stranded RNA is introduced into the cytoplasm of eukaryotic cells, it enters the pathway for the assembly of siRNAs into a RISC complex because it is recognized by Dicer and TRBP (see Figure 1). This process of RNA interference is believed to be an ancient cellular defense against certain viruses and mobile genetic elements in both plants and animals. Plants with mutations in the genes encoding the Dicer and RISC proteins exhibit increased sensitivity to infection by RNA viruses and increased movement of transposons within their genomes. The double-stranded RNA intermediates generated during replication of RNA viruses are thought to be recognized by Dicer, inducing an RNAi response that ultimately degrades the viral mRNAs. During transposition, transposons are inserted into cellular genes in a random orientation, and their transcription from different promoters produces complementary RNAs that can hybridize with each other, initiating the RNAi system, which then interferes with the expression of transposon proteins required for additional transpositions.

In plants and C. elegans, RNA interference can be induced in all cells of the organism by introduction of double-stranded RNA into just a few cells. Such organism-wide induction re quires production of a protein that is homologous to the RNA replicases of RNA viruses. It has been revealed that double stranded siRNAs are replicated and then transferred to other cells in these organisms. In plants, the transfer of siRNAs might occur through plasmodesmata, the cytoplasmic connections between plant cells that traverse the cell walls between them. Organism-wide induction of RNA interference does not occur in Drosophila or mammals, presumably because their genomes do not encode RNA replicase homologs.

In mammalian cells, the introduction of long RNA-RNA duplex molecules into the cytoplasm results in generalized inhibition of protein synthesis via the PKR pathway, discussed further below. This response greatly limits the use of long double-stranded RNAs to experimentally in duce RNA interference against a specific targeted mRNA. Fortunately, researchers discovered that double-stranded siRNAs 21–23 nucleotides long with two-base 3′ single stranded regions lead to the generation of single-stranded RNAs that are incorporated into functional siRNA RISC complexes without inducing the generalized inhibition of protein synthesis. This discovery has allowed researchers to use synthetic double-stranded siRNAs to knock down the expression of specific genes in human cells as well as in other mammals. This siRNA knockdown method is now widely used in studies of diverse processes, including the RNAi pathway itself!

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قسم الشؤون الفكريّة يُهدي إصداراته العلمية إلى مكتبات محافظة كركوك

شعبة فاطمة بنت أسد تطلق مسابقتها الإلكترونية (منارة الجود في مهد الصبا) لكتابة القصة للأطفال

المجمع العلمي يقيم برنامجًا قرآنيًا تعليميًا في النجف الأشرف

قسم الشؤون الدينية يقيم مجلسًا عزائيًّا لاستذكار أهل البيت (عليهم السلام)

المزيد

الآخبار الصحية

ما هي أفضل العصائر لصحة القلب وتقوية المناعة والنوم العميق؟

الأطعمة الغنية بالبروتينات النباتية تقلل خطر الأمراض المزمنة

وجع الأوتار قد يخفي خطرا صحيا مرتبطا بالقلب

أزمة "السفينة الموبوءة"..الأرجنتين تحقق في مصدر تفشي "هانتا"

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الآخبار التكنلوجية

باحثون يحددون تأثير القهوة على الميكروبيوم والمزاج

روسيا.. ابتكار تقنية لمعالجة سبائك التيتانيوم لحماية الطائرات والسيارات

بكين وواشنطن تدرسان إطلاق محادثات رسمية حول الذكاء الاصطناعي

لأول مرة في الولايات المتحدة.. تشغيل مفاعل مصغر نووي يغذي الذكاء الاصطناعي بالطاقة

المزيد

مواضيع ذات صلة

Protein Synthesis Can Be Globally Regulated

Cytoplasmic Polyadenylation Promotes Translation of Some mRNAs

Alternative Polyadenylation Increases miRNA Control Options

Micro-RNAs Repress Translation and Induce Degradation of Specific mRNAs

Degradation of mRNAs in the Cytoplasm Occurs by Several Mechanisms

RNA Processing Solves the Problem of Pervasive Transcription of the Genome in Metazoans

Nuclear Exoribonucleases Degrade RNA That Is Processed Out of Pre-mRNAs

Microchip Electrophoresis

Electrophoresis of RNA

Pulsed-Field Gel Electrophoresis

3′ Cleavage and Polyadenylation of Pre-mRNAs Are Tightly Coupled

Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs