Monday, October 10, 2011

mRNA stability, transport, and turnover

mRNA stability, transport, and turnover


A fundamental regulator of gene expression in all cell types and in all subcellu-lar compartments is the stability of translatable transcripts. In general, mRNAs do not have long half-lives, presumably so as to prevent the over-production of a normal protein which could, in turn, disrupt homeostasis and give rise to a disease state. At the same time, mRNAs must remain stable long enough to become recognized and engaged by the translation apparatus, which are the intrinsic functions of the 5 cap. If large quantities of a protein are to be produced in a cell, one may expect that the corresponding gene will be tran-scribed with a greater frequency than other genes with, for example, house-keeping functions. Similarly, the formation of mRNA secondary structures close to the 5 end in both plants animals can severely limit the scanning of the mRNA such that the initiation of translation is all but inhibited ( Pain, 1996 ; Kozak, 1991 ; Dinesh-Kumar and Miller, 1993 ; Futterer and Hohn, 1996 ; for review, see Kozak, 1999 ). At the other end of the molecule, the length of the poly(A) tail itself plays a role in mRNA stability, as shortening of the poly(A) tail results in destabiliza-tion of cytoplasmic transcripts ( Decker and Parker, 1994 ; Beelman and Parker, 1995 ). Early studies demonstrated that the enzymatic removal of the poly(A) tract from globin mRNA results in a rapid loss of translatability in frog oocytes due to rapid degradation ( Huez et al ., 1974 ; Marbaix et al ., 1975 ). More recent studies have demonstrated the role of the 3 AREs; deletion of these sequences greatly reduces the rate of deadenylation, thereby prolonging mRNA in the cytoplasm ( Wilson and Treisman, 1988 ; Shyu et al ., 1991 ; Decker and Parker, 1993 ; Chen and Shyu, 1994 ). Further, an increasing body of evidence is suggesting that both the length and nucleotide composition of the 5 UTR and 3 UTR play a previously unrecognized role in the stability of the tran-script (reviewed by Lewin, 2008 ). Finally, another recently discovered pathway known as nonsense-mediated mRNA decay appears to be at work in eukaryotic cells which rapidly targets for degradation mRNAs.
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Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.