Cytoplasmic Mechanisms of Posttranscriptional Control

Before proceeding, let's quickly review the steps in gene expression at which control is exerted. We saw in the previous chapter that regulation of transcription initiation is the principal mechanism for controlling expression of genes. In preceding sections of this chapter we learned that transcription of some genes is regulated by controlling the transport into and out of the nucleus of transcription factors that regulate them. We also learned that expression of protein isoforms is controlled by regulating alternative RNA splicing. Although transport of fully processed mRNAs to the cytoplasm rarely is regulated, the transport of unspliced retroviral RNAs is specifically controlled.

In this section we consider other mechanisms of post-transcriptional control that contribute to regulating the expression of some genes. Most of these mechanisms operate in the cytoplasm, controlling the stability or localization of mRNA or its translation into protein. We begin by discussing two recently discovered and related mechanisms of gene control.

Micro RNAs Repress Translation of Specific mRNAs

Micro RNAs (miRNAs) were first discovered during analysis of mutations in the lin-4 and let-7 genes of the nematode C. elegans. Cloning and analysis of wild-type lin-4 and let-7 revealed that they encode no protein products, but rather RNAs only 21 and 22 nucleotides long, respectively, that hybridize to the 3' untranslated regions of specific target mRNAs. For example, the lin-4 miRNA, which is expressed early in embryogenesis, hybridizes to the 3' untranslated regions of both the lin-14 and lin-28 mRNAs, thereby repressing translation of these mRNAs by an as yet unknown mechanism. Expression of lin-4 miRNA ceases later in development, allowing translation of newly synthesized lin-14 and lin-28 mRNAs at that time. Expression of let-7 miRNA occurs at comparable times during embryogenesis of all bilaterally symmetric animals. The role of lin-4 and let-7 miRNAs in coordinating the timing of early developmental events in C. elegans is discussed in Chapter 22. Here we focus on what is currently understood about how miRNAs repress translation.

This form of translational regulation is probably not limited to the lin-4 and let-7 miRNAs in C. elegans; about 100 different miRNAs have been found in C. elegans, and at least as many in humans. All miRNAs appear to be formed by processing of «70-nucleotide precursor RNAs that form hairpin structures with a few base-pair mismatches in the stem of the hairpin. A ribonuclease called Dicer, which cleaves double-stranded RNA, is required for production of miRNAs from these precursors. The base pairing between a miRNA and the 3' untranslated region of its target mRNAs is not precisely complementary, so that some base-pair mismatches occur in the hybridized region. This mismatching distinguishes miRNA-mediated translational repression from the related phenomenon of RNA interference, which we describe next.

RNA Interference Induces Degradation of mRNAs with Sequences Complementary to Double-Stranded RNAs

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, 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 an introduced double-stranded RNA soon was 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 one strand of the double-stranded RNA. Because of the specificity of RNA interference in targeting mRNAs for destruction, it has become a powerful experimental tool for studying gene function (see Figure 9-43).

Subsequent biochemical studies with extracts of Drosophila embryos showed that a long double-stranded RNA that mediates interference is initially processed into a double-stranded intermediate referred to as short interfering RNA (siRNA). The strands in siRNA contain 21-23 nucleotides hybridized to each other so that the two bases at the 3' end of each strand are single-stranded. The finding that Dicer ribonuclease is required for formation of siRNAs suggested that RNA interference and miRNA-mediated translational repression are related processes.

Recent studies indicate that double-stranded siRNAs and miRNAs are further processed into a multiprotein complex containing only one of the RNA strands (Figure 12-27). This RNA-induced silencing complex (RISC) then cleaves target RNAs that are precisely complementary to their corresponding single-stranded siRNAs. These complexes also appear to function in the inhibition of translation by miRNAs. The human let-7 miRNA, for instance, is found in an RNA-induced silencing complex that can cleave a synthetic target RNA that is precisely complementary to let-7 miRNA. However, the same complex does not cleave an RNA whose

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