Rethinking the central dogma: Noncoding RNAs are biologically relevant

Urologic Oncology: Seminars and Original Investigations 27 (2009) 304 –306

Seminar article

Rethinking the central dogma: Noncoding RNAs are biologically relevant Victoria L. Robinson, Ph.D.* Department of Surgery/Section of Urology, The University of Chicago, Chicago, IL 60637, USA

Abstract Non-coding RNAs (ncRNAs) are a large class of functional molecules with over 100 unique classes described to date. ncRNAs are diverse in terms of their function and size. A relatively new class of small ncRNA, called microRNAs (miRNA), have received a great deal of attention in the literature in recent years. miRNAs are endogenously encoded gene families that demonstrate striking evolutionary conservation. miRNAs serve essential and diverse physiological functions such as differentiation and development, proliferation, maintaining cell type phenotypes, and many others. The discovery and ongoing investigation of miRNAs is part of a revolution in biology that is changing the basic concepts of gene expression and RNA functionality. A single miRNA can participate in controlling the expression of up to several hundred protein-coding genes by interacting with mRNAs, generally in 3= untranslated regions. Our new and developing understanding of miRNAs, and other ncRNAs, promises to lead to significant contributions to medicine. Specifically, miRNAs are likely to serve as the basis for novel therapies and diagnostic tools. © 2009 Elsevier Inc. All rights reserved. Keywords: microRNA; Noncoding RNA

Introduction microRNAs (miRNA) are a relatively new class of functional small noncoding RNAs (ncRNA). The discovery and ongoing investigation of miRNAs is part of a revolution in biology that is changing the basic concepts of gene expression and RNA functionality. miRNA are produced from endogenous genes that are located throughout the human genome. Given the abundance and importance of miRNAs, it seems surprising that they were only discovered in the last decade. A probable cause is that biologists are trained to accept the “central dogma of molecular biology.” According to the central dogma, RNA is the intermediary of information flow between the genetic material (DNA) and the end product (protein). DNA allows for long-term storage of the genetic code and is stable, orderly, and inert. The DNA code is transcribed into messenger RNA, which allows for short-term storage and is highly unstable. Proteins, the programs of the cells, are the physical manifestations of the information recorded in a genome. Exceptions to the central dogma include reverse transcriptases, enzymes that synthesize DNA from RNA. Moreover, DNA is not static and does more than serve as an inert source of information. Rather, DNA is dynamic and modifi* Corresponding author. Tel.: ⫹1-773-834-1628; fax: ⫹1-773-702-1001. E-mail address: [email protected]. 1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2008.11.004

able. DNA modifications contribute to gene transcription regulation, such as chromatin organization and remodeling, genomic imprinting, DNA methylation, and other mechanisms. Another likely cause for the delay in miRNA discovery is the faulty “junk DNA hypothesis.” The fact that only a small percentage of the human genome codes for protein has long mystified scientists. For decades it was believed that the majority of our DNA, up to 80% to 90%, served no biological purpose. Rather, this “junk DNA” was believed to have accumulated over time as the result of “freeloaders,” “parasites,” “hitchhikers,” “ancient viral invaders,” and “evolutionary fossils.” In 2001, the human genome project revealed that while only 2% of our DNA is protein-coding, up to 50% is unique sequence, and the remaining 50% is repetitive sequence, mainly transposable elements. While uncovering the purpose of the 1500 megabases of nonprotein-coding unique DNA will likely be ongoing for many years to come, a significant fraction is in fact noncoding RNA (ncRNA) genes. Small ncRNAs are powerful sequence-specific post-transcriptional regulators of gene expression ncRNAs are a large class of functional molecules with over 100 unique classes described to date, and are diverse in terms of their function and size. Well-described functional

V.L. Robinson / Urologic Oncology: Seminars and Original Investigations 27 (2009) 304 –306

ncRNAs include the RNA components of ribosomes, RNA splicing complexes, and telomere maintenance machinery. Two types of small ncRNAs have received a great deal of attention in the literature recently: small interfering RNA (siRNA) and microRNA (miRNA). siRNA and miRNA are similar but distinct in important ways. Among the similarities are their size (⬃20 base pairs) and their ability to suppress expression of genes with related sequences. There is also some overlap in the cell machinery needed for their function. siRNA and miRNA are, however, surprisingly different. siRNAs induce RNA interference (RNAi), a potent, highly specific gene-silencing phenomenon that is initiated by dsRNA [1]. Although endogenous siRNAs exist in viruses and some organisms (not in mammals to date), they are primarily experimental tools. siRNAs suppress gene expression by inducing degradation of mRNAs containing sequence that is a perfect complement to the siRNA, and therefore a given siRNA will theoretically suppress expression of only 1 mRNA. On the other hand, miRNAs are endogenously encoded gene families that demonstrate striking evolutionary conservation. miRNAs serve essential and diverse physiological functions such as differentiation and development, proliferation, maintaining cell type phenotypes, and many others [1]. miRNAs function as posttranscriptional regulators of gene expression through a number of different mechanisms, including promoting RNA degradation (directly and indirectly), repressing translation initiation, blocking protein elongation, and possible additional mechanisms, such as engaging in complexes of sequestered mRNAs [2]. According to miRbase release 12.0, 695 human miRNA genes have been described, although the discovery of new miRNAs is an ongoing process [3]. miRNAs are categorized using a standard nomenclature. The first 3 letters designate the species (hsa for human, mmu for mouse, rno for rat, cel for C. elegans, etc.), and the number indicates the sequence. For example, hsa-miR-1 is identical to cel-miR-1 and nearly identical to rno-miR-1. When a new miRNA is identified, it is first compared with other known miRNAs before a number is assigned. Identical or closely related miRNAs within the same species are given sub-names, usually letters, but sometimes numbers (i.e., hsa-miR-199a/hsa-miR-199b and hsa-miR-138-1/hsa-miR-138-2). Active mature miRNAs, usually 22 nucleotides long in humans, are made through a defined biogenesis pathway. miRNAs are transcribed by RNA polymerase II from unique genes or from introns of protein coding genes into a primary RNA transcript called a pri-miRNA. miRNA are often located closely together, such that they are transcribed as a cluster of 2 or more. Within the pri-miRNAs, a stem-loop forms containing the mature miRNA sequences. Each miRNA gene has the potential to make 2 individual mature miRNA sequences, 1 from each side of the stem loop (not necessarily complementary to each other). The mature miRNA considered to be the less abundant form is designated the star (*) form, although the updated nomenclature designates each mature miRNA as 5p

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(from the 5= side of the step loop) or 3p (from the 3=). The miRNA stem-loop is excised by an RNP complex to form the pre-miRNA, ⬃70 nucleotides long, which is cleaved by Dicer into a short dsRNA. Through a poorly-described mechanism, 1 strand is selected, while the other is degraded or sequestered. The mature single-stranded miRNA, as part of a complex called the RNA-induced silencing complex (miRISC), interacts with partially-complementary sequences found in the 3= UTRs of protein-coding mRNAs [1]. It is not known what factors control the outcome of an interaction between a specific miRNA/mRNA pair, although some species-specific differences have been noted. Initial reports suggested that in mammalian systems, miRNAs primarily prevent efficient translation into protein. However, despite extensive efforts, a direct role in translation has yet to be demonstrated [4]. The striking conservation of miRNA sequences between evolutionarily diverse species supports the notion that miRNAs are critical mediators of biological processes [1]. Experimental data from a variety of organisms have confirmed the importance of miRNAs in development and differentiation, cell-fate decisions, and diverse physiological functions across many organ systems [5,6]. Altered miRNA expression can lead to loss of cell-type identity, a “hallmark” of cancer. As miRNAs are proposed to control the protein levels of up to hundreds of target genes, they are functionally active mediators of potentially every cell signaling pathway and physiological process. Over that last 10 years, the miRNA field has rapidly progressed. The first 2 miRNAs, lin-4 and let-7, were reported in the worm C. elegans in 1998 and 2000, respectively [7,8]. Lin-4 and let-7 were not renamed using the standard nomenclature. A major breakthrough came in 2001, when evidence from several groups revealed that miRNAs are an abundant class of genes in plants and animals, and are conserved in many organisms including humans [9]. A myriad of methods, tools, public databases, and reagents are now available for performing miRNA research. Several computational prediction algorithms were developed to predict the protein-coding target genes of miRNAs. The 3 most commonly used publicly available algorithms are TargetScan [10], PicTar [11], MiRanda [12], each of which typically produces 100 to 400 potential targets for a single miRNA sequence.

miRNAs are critical mediators of tumorigenesis and have shown enormous promise for cancer diagnosis and prognosis A definitive link between alterations in miRNA expression and function has now been established through genomic deletion evidence, transgenic mouse models, and global miRNA profiling [13]. miRNAs are potentially involved in every step of tumorigenesis and can function as both tumor-suppressor genes and oncogenes [14]. Despite

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being discovered very recently, many miRNAs have been directly implicated in cancer. For example, the miR-34 family is transcriptionally up-regulated by p53 and subsequently mediates downstream functions critical to p53 tumor suppressor activity [15,16]. Another example is a cluster of 6 miRNAs on chromosome 13 known as the 17–92 cluster, or oncomir-1, which is expressed as a single primary transcript. Expression of the 17–92 cluster is increased in human cancers and functions as an oncogene in lymphoma and lung cancer animal models [17]. miRNAs have the potential to revolutionize the way cancers are detected and treated. Signatures using fewer than 300 miRNAs are significantly more accurate at classifying tumors compared with mRNA signatures containing over 16,000 mRNAs. miRNA expression profiles, or even the status of only a few miRNA, are better for predicting patient outcomes compared with standard methods [18]. A large body of recent literature supports this concept [19,20]. miRNAs are also attractive targets for novel therapeutic approaches.

RNA: More than just the messenger RNA genes are much more important and diverse in their functions than previously anticipated. In the modern view of gene expression, although proteins are critical for each step, ncRNAs, especially small ncRNAs, perform essential functions. In particular, by fine-tuning gene expression miRNAs are critical players in a vast array of physiological processes. Recent data suggest that miRNA function is alerted in every single human tumor and many other disease states, opening the potential for new miRNA-based clinical applications, such as diagnostic and prognostic tools and therapies that target miRNA function. Our understanding of the importance of ncRNA is still in its infancy. Already, RNAi is undergoing a re-definition process. It now appears that small ncRNAs are capable of wide-ranging functions, possibly activating transcription and participating in heterochromatin silencing. Besides miRNAs, there are additional types of endogenous ncRNAs, including gigaRNA, ultra conserved region RNA, and piwiRNA. A task for the immediate future will be defining the functions of ncRNAs, developing better target prediction tools and comprehensive databases of miRNA sequences, and improving upon our current understanding of mammalian translation. As the expanse of ncRNA functions is uncovered, scientists should

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