17.5 RNA Interference Is an Important Mechanism of Gene Regulation

The expression of a number of eukaryotic genes is controlled through RNA interference, also known as RNA silencing and posttranscriptional gene silencing (see Chapter 14). Research suggests that as much as 30% of human genes are regulated by RNA interference. RNA interference is widespread in eukaryotes, existing in fungi, plants, and animals. This mechanism is also widely used as a powerful technique for artificially regulating gene expression in genetically engineered organisms (see Chapter 19).

Small Interfering RNAs and MicroRNAs

RNA interference is triggered by microRNAs (miRNAs) and small interfering RNAs (siRNAs), depending on their origin and mode of action (see Chapter 14). An enzyme called Dicer cleaves and processes double-stranded RNA to produce single-stranded siRNAs or miRNAs that are from 21 to 25 nucleotides in length (Figure 17.13) and pair with proteins to form an RNA-induced silencing complex (RISC). The RNA component of RISC then pairs with complementary base sequences of specific mRNA molecules, most often with sequences in the 3′ UTR of the mRNA. Small interfering RNAs tend to base pair perfectly with the mRNAs, whereas miRNAs often form less-than-perfect pairings.

Figure 17.13: RNA silencing leads to the degradation of mRNA or to the inhibition of translation or transcription. (a) Small interfering RNAs (siRNAs) degrade mRNA by cleavage. (b) MicroRNAs (miRNAs) lead to the inhibition of translation. (c) Some small interfering RNAs (siRNAs) methylate histone proteins or DNA, inhibiting transcription.

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Mechanisms of Gene Regulation by RNA Interference

Small interfering RNAs and microRNAs regulate gene expression through at least four distinct mechanisms: (1) cleavage of mRNA, (2) inhibition of translation, (3) transcriptional silencing, or (4) degradation of mRNA.

RNA Cleavage

RISCs that contain an siRNA (and some that contain an miRNA) pair with mRNA molecules and cleave the mRNA near the middle of the bound siRNA (see Figure 17.13a). This cleavage is carried out by a protein that is sometimes referred to as “Slicer.” After cleavage, the mRNA is further degraded. Thus, the presence of siRNAs and miRNAs increase the rate at which mRNAs are broken down and decrease the amount of protein produced.

Inhibition of Translation

Some miRNAs regulate genes by inhibiting the translation of their complementary mRNAs (see Figure 17.13b). For example, an important gene in flower development in Arabidopsis thaliana is APETALA2. The expression of this gene is regulated by an miRNA that base pairs with nucleotides in the coding region of APETALA2 mRNA and inhibits its translation.

The exact mechanism by which miRNAs repress translation is not known, but some research suggests that it can inhibit both the initiation step of translation and steps after translation initiation such as those causing premature termination. Many mRNAs have multiple miRNA-binding sites, and translation is most efficiently inhibited when multiple miRNAs are bound to the mRNA.

Transcriptional Silencing

Other siRNAs silence transcription by altering chromatin structure. These siRNAs combine with proteins to form a complex called RITS (for RNA transcriptional silencing; see Figure 17.13c), which is analogous to RISC. The siRNA component of RITS then binds to its complementary sequence in DNA or an RNA molecule in the process of being transcribed and represses transcription by attracting enzymes that methylate the tails of histone proteins. The addition of methyl groups to the histones causes them to bind DNA more tightly, restricting the access of proteins and enzymes necessary to carry out transcription (see earlier section on histone modification). Some miRNAs bind to complementary sequences in DNA and attract enzymes that methylate the DNA directly, which also leads to the suppression of transcription (see earlier section on DNA methylation).

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Slicer-Independent Degradation of mRNA

A final mechanism by which miRNAs regulate gene expression is by triggering the decay of mRNA in a process that does not require Slicer activity. For example, a short-lived mRNA with an AU-rich element in its 3′ UTR is degraded by an RNA-silencing mechanism. Researchers have identified an miRNA with a sequence that is complementary to the consensus sequence in the AU-rich element. This miRNA binds to the AU-rich element and, in a way that is not yet fully understood, brings about the degradation of the mRNA in a process that requires Dicer and RISC.

The Control of Development by RNA Interference

Much of development in multicellular eukaryotes is through gene regulation: different genes are turned on and off at specific times (see Chapter 22). In fact, when miRNAs were first discovered, researchers noticed that a mutation in an miRNA in C. elegans caused a developmental defect. Research now demonstrates that miRNA molecules are key factors in controlling development in plants, animals, and humans. For example, the vertebrate heart develops through the programmed differentiation and proliferation of cardiomyocytes, which are controlled by a specific miRNA termed miR-1-1.

Recent studies demonstrate that, through their effects on gene expression, miRNAs play important roles in many diseases and disorders. For example, a genetic form of hearing loss has been associated with a mutation in the gene that encodes an miRNA. Other miRNAs are associated with heart disease. One miRNA called miR-1-2 is highly expressed in heart muscle. Mice genetically engineered to express only 50% of the normal amount of miR-1-2 frequently have holes in the wall that separates their left and right ventricles, a common congenital heart defect seen in newborn humans. Overexpression of another miRNA called miR-1 in the hearts of adult mice causes cardiac arrhythmia—irregular electrical activity of the heart that can be life-threatening in humans. Changes in the expression of other miRNAs have been associated with cancer.

CONCEPTS

RNA silencing is initiated by double-stranded RNA molecules that are cleaved and processed. The resulting siRNAs or miRNAs combine with proteins to form complexes that bind to complementary sequences in mRNA or DNA. The siRNAs and miRNAs affect gene expression by cleaving mRNA, inhibiting translation, altering chromatin structure, or triggering RNA degradation.

CONCEPT CHECK 5

In RNA silencing, siRNAs and miRNAs usually bind to which part of the mRNA molecules that they control?

  1. 5′ UTR.
  2. Segment that encodes amino acids.
  3. 3′ poly(A) tail.
  4. 3′ UTR.