Another powerful mechanism of regulation of gene expression at the RNA level is RNA interference (RNAi), mediated by miRNAs; miR NAs are small RNAs (21 to 24 nucleotides in length) that bind to complementary sequences on target mRNA transcripts, prevalently in the 3′UTR. This binding results in transcript degradation and inhibition of translation and consequent silencing of gene expression (i.e., lack of protein output). There are now more than 2000 miRNA molecules coded in the human genome that regulate approximately one third of human genes. Each miRNA has the potential to target hundreds of genes. Conversely, an estimated 60% of all mRNAs have one or more sequences that are predicted to interact with miR NAs. RNAi has been very useful in the laboratory, allowing investigators to repress the expression of specific genes to study artificially induced phenotypes. In these studies, siRNAs are synthesized to bind to homologous sequences within specific mRNAs. The siRNAs and respective nontargeting controls are then transfected into cells, where they mediate destruction of their target mRNA through endogenous ribonucleases. Repression of gene expression in this manner has become known as “gene knockdown” and continues to be widely used to define the function of genes, by assessing what function the cell lacks in the absence of the expression of the target gene. The siRNA-mediated knockdown strategy is now being partially replaced by so-called CRISPR/Cas9-mediated gene deletion, which is covered in other chapters of this book.

To perform their function, both miRNAs and siRNAs are bound by a multiprotein complex called the RNA-induced silencing com plex (RISC). Members of the Ago family of proteins are central to RISC function. Argonautes contain two conserved RNA-binding domains: a PAZ domain that can bind the single-stranded 3′ end of the mature miRNA and a PIWI domain that functions to interact with the 5′ end of the guide strand. Ago proteins bind the mature miRNA, orient it for interaction with a target mRNA, and may also directly cleave target transcripts (Ago2). The specificity of miRNA and siRNA interactions with their target mRNAs mediates how they regulate gene expression. For example, the specificity of miRNA targeting is ruled by Watson-Crick complementarities between positions 2 to 7 at the 5′ end of the miRNA, the so-called seed sequence, and the 3′ UTR of their target mRNAs.

Two models have been proposed to explain how miRNAs and siRNAs interfere with the expression of target genes: directed degradation of the target mRNA, performed as aforementioned by the RISC complex, and interference with the translation of a target mRNA (Fig. 1). In the case of directed mRNA degradation, the proposed model involves miRNA-mRNA binding and recruitment of RISC, which ultimately leads to degradation of the target mRNA. In the interference model the interaction of miRNA, RISC, and mRNA blocks the ribosomal machinery along the mRNA transcript, preventing translation. In reality, both models could contribute to gene expression downregulation. Most of the proteins necessary for miRNA-mediated gene silencing are located in cytoplasmic organelles, so called P-bodies, that are formed via phase separation. The specific circumstance of P-body involvement in miRNA-mediated RNA degradation is still an area of active research.

Fig1. RNA INTERFERENCE AND THE CONTROL OF TET2 GENE EXPRESSION. The fully processed miR-22 microRNA associates with Ago proteins and binds target messenger RNAs (mRNAs), such as the 3′ untranslated region of Tet2, by sequence complementarity. RNA interference results in decreased translation and increased degradation of the target mRNA. The expression of several miRNAs, such as miR-22, is altered in hematologic malignancies, leading to dysregulation of all their targets. MDSs, Myelodysplastic syndromes.

Various diseases, including many cancers, exhibit aberrant expression of miRNAs.[1] Regulation of the Tet2 mRNA by miR-22, dysregulated in MDSs and AML, is shown in Fig. 1. Another example is the miR-15a/miR-16-1 cluster (located on chromosome 13q) chronic lymphocytic leukemia (CLL). When this cluster is deleted in B lymphocytes, there are higher levels of antiapoptotic proteins such as BCL2 and MCL1 but also higher levels of the tumor suppressor protein TP53. High levels of antiapoptosis yet with an intact TP53 tumor suppressor pathway could explain why 13q deletions in CLL are associated with an indolent form of the disease.[2] Several miRNAs have been identified that regulate hematopoiesis and in particular lineage choice and differentiation. One such miRNA is miR-155 that retains HSCs and progenitor cells in an undifferentiated state and is downregulated with lineage differentiation. MiR-155 is regulated by the homeobox gene HOXA9.

References

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[1] Shah MY, Calin GA. MicroRNAs as therapeutic targets in human cancers. Wiley Interdiscip Rev RNA. 2014;5(4):537–548.

[2] Calin GA, Pekarsky Y, Croce CM. The role of microRNA and other noncoding RNA in the pathogenesis of chronic lymphocytic leukemia. Best Pract Res Clin Haematol. 2007;20(3):425–437.

مواضيع ذات صلة

RNA Granules and Membraneless Organelles

Balancing Synthesis of Ribosomal Components

Regulation of Ribosome Synthesis

Proportionality of Ribosome Levels and Growth Rates

The Signal Recognition Particle and Translocation

Verifying the Signal Peptide Model

Directing Proteins to Specific Cellular Sites

Protein Elongation Rates

Messenger Instability

موقع الانتساخ ومتطلباته

Resolution of a Paradox in Protein Synthesis

Chaperones and Catalyzed Protein Folding