The biology of RNA is quite intriguing. A contemporary discussion and thinking on the plethora of diversity they show in terms of structure, function and complexity demands understanding of the molecule in a blue sky perspective. The multiplicity of their forms from being coding, non-coding and their proven role in therapeutics makes the small molecule pivotal thus opening a new world of ribomics or ribonucleomics.
Ribonucleic acid (RNA) is a biomolecule which consists of ordered repeats of nucleotide units. The backbone cocktail is a mixture of nitrogenous base, ribose sugar and phosphate moiety. RNA is quite similar to DNA but differs on the parameters of being single stranded, harbouring a ribose sugar and thymine being replaced by uracil. DNA undergoes transcription to yield RNA the process mediated by RNA polymerases which is further translated to yield the building blocks of life-proteins. A fine orchestra of RNA and protein assembly is involved to produce life’s key molecular machine, ribosomes the site for protein synthesis, which is nature’s marvellous ergonomic design. Diversity exists in forms, structure and function of RNA and their crucial role in fine tuning and regulating intricate gene expression and being the genome material of viruses has evoked great interest globally to unveil these molecules. Thus RNA biology takes centre stage of today’s therapeutic scenario.
Types and diversity of RNAs
The diversity of RNA is mind boggling with pleotropicity in their functional assignments. The family is mainly represented by coding and non coding RNAs. The transcriptomal process involves a consortium of four major RNAs the hnRNA (hetronuclear) being processed further to yield m-RNA by polyadenylation at the 3’ tail and capping at 5’ end this forms the coding RNAs. All other RNA falls under the broad umbrella of non- coding RNA. The rRNA (ribosomal) and proteins combine together to form macromolecular machines called ribosomes which serve as site for protein synthesis. The tRNAs are the carrier and forwarding agents which read the code (codon —anticodon matching) and bring about one aminoacid from the cellular pool to be attached each time in the elongating chain of polypeptides. The cell harbours twenty different types of tRNAs which are appropriately recruited to form diverse protein molecules.
The non coding RNAs form an even more interesting group of small RNAs. The members have quite interesting functional attributes. The siRNA christened as small interfering RNA or silencing RNA is a class of 20-25 nucleotides, double stranded RNA molecules which are mainly involved in RNAi (RNA interference) pathway. The molecule is involved in PTGS (Post Transcriptional Gene Silencing), antiviral mechanism and chroma in remodelling and dynamics. The enigma is now being investigated in greater details for discovery of the phenomenon by David Baulcombe’s in plants and Thomas Tuschl in mammalian cells which has opened up new vistas in biomedical research and drug development and paved a new branch of interference biology.
Micro RNA (mi RNA) is another member belonging to gene regulatory small RNAs. These 21-23 nucleotide species are synthesised and processed from single stranded RNA precursors and show partial complementarity to m-RNA target molecule. These interesting molecules have multiple function with their role in cellular growth, apoptosis, neuronal remodelling, their enhanced presence may lead to Fragile X mental retardation. They have been further attributed to cancer. These undergo various processing to form a dismantling machine that finally chew up mRNA molecules thus regulating and sometime disorienting normal gene expression. Small nucleolar RNA (sno RNA) or sn RNA (small nuclear RNA) are mainly responsible for bringing out biochemical modification of such as methylation and pseudouridylation of rRNA, tRNA and other small nuclear RNA.
There are some other types of RNAs such as telomerase RNA which provides RNA template that is acted upon by telomerase, a reverse transcriptase to synthesize DNA at the chromosomal ends as it gets shortened up in each replication cycle. The telomerase has role in aging and cancer and also in cri-du chat syndrome. RNase MRP RNA is a subunit component of mitochondrial RNA processing (MRP) enzyme complex. This enzyme is involved in multiple cellular RNA processing and is associated with cartilage-hair hypoplasia (CHH), a recessively inherited developmental disorder. Ribonuclease P (RNase P) is another type of catalytic RNA (Ribozymes) discovered by Sidney Altman (Nobel Prize in Chemistry 1989)-- it cleaves extra precursor RNA from tRNA molecules. It’s further attributed to efficiently control transcription of small non coding RNA genes. Vault RNA (vRNA) is vault ribonucleoprotein complex constituted of major vault protein (MVP) and two minor vault proteins (VPARP and TEPI) with small untranslated RNA molecules-- these are mainly attributed to drug resistance. The YRNA are part of ribonucleoprotein particle (Ro RNP) first identified by Learner et.al as target of autoimmune antibodies in systemic lupus erythmatosus (SLE). Its main function is to put a quality check on maturing 5S rRNA and is proved to be required for DNA replication Piwi-interacting RNA (pi RNA) is the largest class of small RNA molecules expressed in animal cells. They form riboprotein complex on interacting with piwi protein. They are crucial in transcriptional gene silencing of retrotransposons mainly in germ line cells. They are very unique from other family by virtue of their complexity, no sequence conservation and being 26-31 nucleotides in length.
The RNA viruses or retroviruses have RNA as their genetic material. There biology opens an entirely new and exciting field in virology and pathogenesis that is RNA toxicity.
Thus existence of a galaxy of RNA, sharing the common backbone structure but markedly differing in the functional arena controlling process of gene regulation, protein synthesis silencing genes, regulating chromatin dynamics and acting as a quality control machinery, all add to sea of avenues for harnessing their therapeutic potential and thus opening an exciting odyssey for candid evaluation of the molecule in fathomic details.
RNA and disease
The transcriptome comprises of coding as well as significant regions of non coding RNA. The non coding sequences were earlier thought to be junk and no functional attribute was associated with them but with the advancement in trascriptomics they were known to influence and fine tune gene regulation. Expansion of the microsatellite repeats in the non coding regions resulted in the synthesis of pathogenic RNA’s now thought to be the culprit behind some dominantly inherited neurological disorders. The gain of function effects by these non coding regions can be attributed to different pathological consequences. Expression of the toxic RNA is associated with formation of nuclear inclusions and late-onset degenerative changes in brain, heart or skeletal muscle. Myotonic dystrophy is caused by one such phenomenon where regulation of alternative splicing gets compromised due to sequestering of RNA binding proteins by toxic RNAs.
The finest example of toxic role of RNA is polyglutamine disease. In this case dangerous molecular shapes, that resulted by formation of hairpins in case of long CAG repeats were efficiently examined by altering the sequence CAACAG so that no more in could be formed but the sanctity of protein under question and the blueprint of it remainunpertuerbed. It was found that this very alteration drastically reduces neurodegeneration by scrambling the RNA structure mitigated toxicity. This common theme of triple repeat expansion diseases is also prevalent in fragile x syndrome and myotonic dystrophy.
Myotonic Dystrophy (DM) in which either a CTG or CCTG expansion, located within noncoding regions of separate genes, results in strikingly similar effects. The role for an RNA gain-of-function has been firmly established as a major pathogenic event in DM. There is now substantial evidence that other diseases caused by non coding expansions involve an RNA gain-of-function mechanism. These diseases include fragile X tremor ataxia syndrome (FXTAS), spinocerebellar ataxia type 8 (SCA8), SCAb, SCA12, and Huntington’s disease like 2 (HDL2). Recent progress in DM has provided a paradigm for understanding pathogenic mechanisms of RNA mediated disorders.
Fragile X Syndrome (FXS) is caused by expansion of trinucleotide gene sequence CGG on X chromosome. This resulted in inability to express FMR-l protein which is crucial for neuronal development. It’s an inherited mental impairment.
The diseases which are based on toxic RNA are governed by common mechanism of gain of function effect. An insight into the disorders that are being governed by toxic RNA becomes crucial due to absence and nearly no concrete therapeutic regimen against them.
New approaches of RNA
The Human Genome Project delineated about 34,000 genes that code directly for functional proteins. Rest of the genome has been labelled as “junk” because of no obvious function. Recently, RNA biology received global attention with a paradigm shift that the junk genome produces around half a million varieties of RNA which must be having regulatory roles rather than an evolutionary burden. This has open new vistas in research with discussions centered on staggering variety of RNA types produced from this ‘junk” and the huge potential implications that the finding promises. Specific genes associated with diseases processes can be targeted using RNA interference (RNAi). This innovative approach has great therapeutic implications, thus this very idea of harnessing the process as a therapeutic product will herald a new dimension in gene therapy and nucleic acid based therapeutics.
The technology has been a boon since the first report came in 2001 regarding RNA mediated silencing of respiratory syncytial virus (RSV). The technology has undergone several studies and promises attractive alternative in case of hepatitis B and C virus (HBV and HCV, respectively) including dengue virus (DENy), Japanese encephalitis virus (JEV), yellow fever virus (YFV) and West Nile virus (WNV), recent report regarding HIV-1 reveals that targeting the chemokine receptor CCR5 host protein that act as coreceptor for the virus but whose mutation is compatible with normal life can be used for attenuation.
Apart from viral diseases, protozoans that particularly cause havoc have also been shown to get attenuated and silenced. In Trypanosoma bruceI dsRNA could induce sequence-specific m-RNA degradation. The study in case of plasmodium revealed that the mechanisms of RNAi like silencing do exist in plasmodium.
But still dilemma exists as unlike T.brucei, P. falciparum has no relevant homolog to Dicer, Piwi, PAZ or other genes involved in the RNAi pathways. The studies also become quite pivotal due to widespread resistance against currently available antimalarials.
Mycobacterium tuberculosis the world’s most successful pathogen which does evade almost all available chemotherapy by its multidrug resistance has shown promising results in feasibility of utilizing antisense technology. One group has shown that when phosphorothioate-modified antisense oligodeoxyribonucleotides were used against the m-RNA of glutamine synthetase associated with Mycobacterium pathogenicity and formation of a poly-L-glutamate/glutamine cell wall structure, it reduces the expression and activity thereby having profound impact on bacterial replication. Another recent study reports inhibition of mycobacterial growth by inhibition of the lysosomal enzyme betaexosaminidase, which is a peptidoglycan hydrolase that facilitates mycobacteria-induced secretion of lysosomes at the macrophage plasma membrane.
Thymine production is controlled by DHFR (Dihydro Folate Reductase) which is very important for rapidly dividing cells. Inhibiting DHFR will prevent the growth of neoplastic cancerous cells from ordinary cells that do get transformed in to cancerous cells as in prostate cancer (Dr Alexandre Akoulitchev, University of Oxford). Thus an RNA swith could efficiently turn off cancer.
Thus, a birds eye view of the landscape that the technology promises are virtually interesting because it provides an upper hand to silence toxic genes by using an endogenous mechanism that is inherently present in most of the organisms. Be it viral diseases or protozoan or dreaded diseases like tuberculosis and cancer all these can be visualised to be manipulated by this RNA based therapeutics.
RNA and future
This small molecule has been the pool of panacea for treatment of malignant diseases and rescue from other wide variety of old and emerging diseases. The current generation of targeted therapy, however, is not amicable to many new therapeutic targets and increasing drug resistance among patients which add to the burgeoning severity. The new school of thoughts now clearly put forth the importance of genome based safe therapeutics including the RNA technology. Antisense armamentorium when amalgamated with the advance nanoscience and drug delivery strategy is surely bound to fulfil the long cherished dream of biochemists to device “magic bullets” for treatment of whole spectrum of diseases including cancer and AIDS. Recent reports suggest that RNA itself can be ergonomically prototyped to dock many therapeutic molecules simultaneously and target it to particular cell type. The fact that microRNA (miRNA) are evolutionarily conserved, suggests that miRNA therapeutics may have fewer side effects as compared to the artificial siRNAs. At present the miRNAJRNA-i therapeutics field is in its juvenile stage but a wave of optimism exists in the science fraternity to end up the gestation and prepare a firm platform for the birth of RNA therapeutics.
Authors are working with Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow
Courtesy: CDRI – Current R&D Highlights, April-June 2009