Small RNAs in development and disease

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are classes of regulatory small RNA molecules, ranging from 18 to 24 nucleotides in length, whose roles in development and disease are becoming increasingly recognized. They function by altering the stability or translational efficiency of messenger RNAs (mRNAs) with which they share sequence complementarity, and are predicted to affect up to one-third of all human genes. Computer algorithms and microarray data estimate the presence of nearly 1000 human miRNAs, and direct examination of candidate miRNAs has validated their involvement in various cancers, disorders of neuronal development, cardiac hypertrophy, and skin diseases such as psoriasis. This article reviews the history of miRNA and siRNA discovery, key aspects of their biogenesis and mechanism of action, and known connections to human health, with an emphasis on their roles in skin development and disease.

Learning objectives

After completing this learning activity, participants should be able to summarize the relevance of microRNAs in development and disease, explain the molecular steps of how small RNAs regulate their targets within the human cell, and discuss the role of small RNAs in the diagnosis and treatment of disease.

Introduction

The human genome contains about 25,000 genes.¹ The expression of each of these individual genes needs to be appropriately controlled to suit the function and environment of each cell, and must change to respond to new conditions or signals. One of the general aims of biologic research is to understand how a cell's pattern of gene expression is orchestrated to promote coordinated growth and development and to understand how the inappropriate expression of genes is involved with disease.

The basic dogma of molecular biology (Fig 1, A) states that the template for genetic information is encoded in DNA. Genes consist of segments of DNA that are transcribed into RNA molecules, which are then transported from the nucleus to the cytoplasm, where they are translated into proteins. A detailed discussion of this process has been recently published.²

The activity of genes is controlled on each level of this pathway (Fig 1, B). At the DNA level, cells have many ways to regulate how rapidly genes are transcribed. One form of regulation is structural, in which regions of DNA are made more or less physically accessible to the cellular machinery that is required for transcription. Within the nucleus, DNA is packaged among proteins known as histones, which are responsible for organizing DNA. Histones have “tails” that extend outwards from the central part of the protein and contain amino acid sequences that can be chemically modified by the cell. These modifications modulate how tightly the histones are packaged and influence the accessibility of the DNA segments that are associated with them. When they are more accessible, genes can be more easily reached by transcription factors and can be transcribed in higher quantities. Conversely, genes can be kept inactive by being packaged more tightly, keeping them in an inaccessible state.

In addition to structural changes, another mechanism to control gene transcription is to alter the availability, quantity, or activity of transcription factors themselves. A transcription factor is a protein that recognizes a specific sequence in the regulatory region of a gene and influences the rate at which the gene is transcribed. Activity of transcription factors are regulated in many different ways: they can themselves be produced in higher or lower levels; they may be activated or deactivated by chemical modifications such as phosphorylation; their localization to the nucleus can be regulated, such as by binding to a ligand; or their activity can be enhanced or inhibited by interaction with other cofactors and transcription factors. Taken together, there are numerous levels of gene regulation at the DNA level.

Gene regulation also occurs on the level of the RNA transcript. Within the cell, RNA molecules are constantly being created and broken down. Every RNA has a different longevity, depending on features such as the length of the polyadenylation tail and the presence of sequence elements in the regulatory region of the transcript. These features influence the interaction of each RNA with cellular degradation machinery and, by inference, are thought to control the amount of corresponding protein in the cell. Recent experiments indicate that the modulation of RNA stability may be a common global mechanism for cells to control gene activity. Genome-wide analyses suggest that up to 50% of all RNAs undergo significant changes to their stability in response to cellular signals.3, 4 These observations, together with the discovery that microRNAs (miRNAs) function at this level to alter target RNA stability, has sparked a growing interest to further understand the role of RNA turnover in the control of gene action.

Finally, genes are extensively regulated on the protein level. Proteins can have active or inactive conformations, or can require chemical modifications or cofactors to be functionally active. This allows a cell to control where and when a protein is active and to allow a rapid change of a protein to a functional or nonfunctional state. In addition, cellular levels of proteins can be downregulated by targeted breakdown. A well known mechanism to selectively degrade proteins in the cell is through the ubiquitin–proteasome pathway, a process in which a small signal molecule (called “ubiquitin” because of its presence in all eukaryotes) is tagged onto designated proteins, marking them for transport to the proteasome, a barrel-shaped cellular machine that breaks ubiquitinated proteins into peptides and amino acids.

Viewed together, the activity of genes can be regulated on every level, from DNA to RNA to protein. In many instances, the control of any single gene may occur on several different levels, with adjustments being made constantly to change the levels of gene activity to an appropriate state. Despite the many varied mechanisms of gene control that are known, however, historical attention has focused on the regulatory functions of DNA and proteins. RNA has been largely envisioned as an intermediate molecule between the two, with specialized roles in splicing and translation. In this context, it was a revolutionary concept to discover the involvement of RNAs—that were of a surprisingly small size—in the extensive regulation of gene activity in humans. Our understanding of RNA-mediated regulation grows almost daily, as new studies showcase the deep and far-reaching effects of small RNAs on mammalian development and disease. Because of their small size, the very first of these regulatory small RNAs to be discovered were called miRNAs.

MiRNAs were first discovered in the early 1990s in laboratories studying the genetics of development in the roundworm Caenorhabditis elegans. Mutations in two separate C elegans genes, named lin-4 and lin-14, resulted in similar phenotypes in which the worms failed to mature and differentiate properly.⁵ Further characterization of lin-4 uncovered the surprising discovery that the gene itself was a 22-nucleotide (nt) RNA molecule that had multiple sites of sequence complementarity in the lin-14 3′-untranslated region (UTR).6, 7 A molecular model was proposed in which the translation of the lin-14 messenger RNA (mRNA) into a protein was inhibited by the binding of the small lin-4 RNA molecule to the lin-14 3′-UTR.

The discovery of the lin-4 miRNA as a controller of developmental regulation was followed by the identification of another miRNA involved in developmental control, named let-7.⁸ One notable feature of let-7 was that the gene was conserved from the roundworm to other animal species, including humans. This discovery supported the idea that miRNAs might also have important functions among many different species. The use of cloning techniques and computer prediction algorithms, which identify miRNAs within the human genome based on their predicted structures, have now uncovered hundreds of miRNAs appearing in species such as the fruit fly, mouse, and human. Public databases of miRNAs are being constantly updated as investigators identify and elucidate the functions of individual miRNAs (see Britain's Sanger Institute database at http://microrna.sanger.ac.uk/).

Researchers in the field estimate that there are as many as 1000 miRNAs in the human genome,⁹ and that these miRNAs may target up to one-third of all human genes.¹⁰ Using microarray technology, which allows for the characterization of global miRNA expression patterns, the “miRNome” of normal and diseased tissue is becoming rapidly decoded. The identification and cataloguing of miRNAs has progressed much more rapidly than our detailed understanding of their individual functions. Nonetheless, the past few years have begun to reveal critical roles for miRNAs in human development and disease.

In the late 1990s, a landmark study demonstrated that gene expression could be inhibited by the introduction of double-stranded RNA with sequence complementarity to the gene being targeted, a mechanism that was named RNA interference (RNAi).¹¹ The importance of this work would be recognized by the 2006 Nobel Prize in Physiology or Medicine. Biochemical studies revealed that when long, double-stranded RNAs are introduced into a cell, they become diced into short, double-stranded, 21-nt RNAs containing 2-nt 3′ overhangs, known as short interfering RNA (siRNA). The siRNA then guide cellular machinery to target and degrade mRNAs with a similar sequence.

It was recognized that the small RNAs involved in RNAi and the ultimate effect of gene inhibition had similarities to how miRNAs inhibited mRNA protein translation. Our understanding of miRNAs has been helped in part from the parallel study of siRNAs. In particular, these two classes of small RNAs share biochemical machinery involved with their generation and processing.

MicroRNAs originate from all parts of the human genome, including the introns of protein-coding genes, as a cluster with other miRNAs, or as stand-alone genes. Like protein-coding genes, they are activated by transcription factors and are transcribed by RNA polymerase II. A single miRNA has the ability to regulate multiple targets. On the other hand, multiple different miRNAs can also converge and regulate the same target. Taken together, miRNAs form an interconnected regulatory network that does not simply turn genes on or off, but are thought to “tune” the expression level of their target genes.

MiRNA processing has been reviewed in detail,¹² and only the most critical steps and molecular players are discussed here (Fig 2, A). First, the miRNA is transcribed from the genome as a primary RNA transcript (pri-miRNA), which can be many kilobases long. The RNA nucleotides within the pri-miRNA transcript interact with each other to form complementary pairs, causing the RNA to form secondary structures. The most important secondary structure is known as the stem-loop—also known as the hairpin (Fig 2, B)—which is generated when two complementary stretches of RNA base-pair to form the “stem,” with an intervening set of RNA nucleotides that do not interact, which become the circular “loop.” These stretches of paired RNA can occasionally have intervening mismatches, which form a bubble (Fig 2, B).

The intramolecular hairpin structures are recognized by a protein complex in the nucleus that includes the enzyme Drosha, whose function is to excise the hairpin structures from the primary transcript. The excised ∼60 nt hairpins are then called precursor microRNAs (pre-miRNAs), and contain a loop, a 22-nt stretch of complementary double-stranded RNA, and a 2-nt overhang at the 3′ end. Pre-miRNAs are recognized by a protein, exportin-5, that brings them from the nucleus into the cytoplasm.

In the cytoplasm, pre-miRNAs are further processed by an enzyme called Dicer, which removes the loop structure, leaving a double-stranded RNA duplex (miRNA:miRNA∗) that is 22-nt in length. The processed miRNA duplex is taken up by a protein complex known as the RNA-induced silencing complex (RISC), inside which the two RNA strands of the duplex become separated. One strand is used as a mature “guide” strand (miRNA), which will remain bound to RISC and will be the element that recognizes target RNAs. The other strand, known as miRNA∗, becomes dissociated and degraded.

The inhibition of target RNAs occurs through two different mechanisms, depending on the degree with which the guide miRNA strand matches the target RNA. If the pairing between the guide miRNA and the target is imperfect—that is, if there are a significant number of mismatched pairs between the guide miRNA and the 3′-UTR of the target mRNA—then the RISC complex will inhibit the protein translation of that RNA. On the other hand, if there is a perfect match between the guide miRNA and the target RNA, then the target mRNA will be cleaved. One of the important components of RISC is Argonaute (Ago), a family of proteins which bind small RNAs. The Ago2 subtype, which is incorporated into human miRNA and siRNA RISC complexes, is responsible for cleavage of target RNAs.¹³

The components of the miRNA pathway overlap with those involved with processing siRNAs. When double-stranded RNAs are introduced experimentally into cells, Dicer cleaves them into 22-nt siRNAs. These siRNAs are then loaded into RISC in a similar manner as miRNAs (Fig 2, A), with the guide siRNA strand directing the inhibition of the target RNA. miRNAs in humans often do not match perfectly with their targets, and therefore lead to translational inhibition. By contrast, experimental siRNAs are often designed with perfect complementarity to their targets, leading to RNA degradation. The ability of the RISC to perform these different functions is not fully understood, but may depend in part on the subtype of Ago that is incorporated into the RISC complex. For example, although miRNAs and siRNAs in humans can associate with any of the four Ago subtypes, only Ago2 is capable of RNA cleavage.¹⁴

Despite the similarities between miRNAs and siRNAs, the two classes of small RNAs have several key differences, both in definition and concept. MiRNAs refer to small RNAs produced naturally from the human genome, and have diverse and widespread roles. They are generated by transcribing a single RNA that forms an intramolecular hairpin intermediate during processing. As stated before, miRNAs in humans most often have imperfect complementarity to their targets, leading to translational inhibition.

By contrast, siRNAs can be either exogenous or endogenous—that is, either naturally occurring in the genome or introduced from outside the cell. Endogenous siRNAs are created from two separate but complementary transcripts—for example, from bidirectional transcription at the same locus—resulting in a long, perfectly-matched duplex that is subsequently diced into siRNAs. The presence and roles of endogenous siRNAs in humans are still being discovered; thus far, they have been associated with providing a defense against viral infections15, 16 and in the protection against activation of mobile genomic elements.¹⁷

The recent attention towards small RNAs has fueled the discovery of a number of new small RNA classes, each of which are currently classified into different groups based on their different functions or mechanisms by which they are generated. These include trans-acting siRNAs (tasiRNA), a type of small RNA observed only in plants to date¹⁸; repeat-associated siRNAs (rasiRNA), a group of small RNAs that are produced independent of Dicer and Ago, and are thought to protect genome stability¹⁹; and Piwi-interacting RNAs (piRNAs), a set of small RNAs described in the mouse and rat testes that have been shown to have an essential role in the viability of germline stem cells.²⁰ The recent and rapid discovery of these small RNAs reflects the current excitement in the field, and suggests the possibility that small RNAs may be central to other cellular processes that are still unknown. The remainder of this review will focus only on miRNAs, whose impact is better understood at the current time, and the use of siRNAs in the context of laboratory research and disease therapy.