2D-PAGE as an effective method of RNA degradome analysis

The continuously growing interest in small regulatory RNA exploration is one of the important factors that have inspired the recent development of new high throughput techniques such as DNA microarrays or next generation sequencing. Each of these methods offers some significant advantages but at the same time each of them is expensive, laborious and challenging especially in terms of data analysis. Therefore, there is still a need to develop new analytical methods enabling the fast, simple and cost-effective examination of the complex RNA mixtures. Recently, increasing attention has been focused on the RNA degradome as a potential source of riboregulators. Accordingly, we attempted to employ a two-dimensional gel electrophoresis as a quick and uncomplicated method of profiling RNA degradome in plant or human cells. This technique has been successfully used in proteome analysis. However, its application in nucleic acids studies has been very limited. Here we demonstrate that two dimensional electrophoresis is a technique which allows one to quickly and cost-effectively identify and compare the profiles of 10–90 nucleotide long RNA accumulation in various cells and organs

Impact of RNA degradation on gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.</p> <p>Methods</p> <p>To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.</p> <p>Results</p> <p>The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.</p> <p>Conclusions</p> <p>Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.</p

Three Essential Ribonucleases—RNase Y, J1, and III—Control the Abundance of a Majority of Bacillus subtilis mRNAs

Bacillus subtilis possesses three essential enzymes thought to be involved in mRNA decay to varying degrees, namely RNase Y, RNase J1, and RNase III. Using recently developed high-resolution tiling arrays, we examined the effect of depletion of each of these enzymes on RNA abundance over the whole genome. The data are consistent with a model in which the degradation of a significant number of transcripts is dependent on endonucleolytic cleavage by RNase Y, followed by degradation of the downstream fragment by the 5′–3′ exoribonuclease RNase J1. However, many full-size transcripts also accumulate under conditions of RNase J1 insufficiency, compatible with a model whereby RNase J1 degrades transcripts either directly from the 5′ end or very close to it. Although the abundance of a large number of transcripts was altered by depletion of RNase III, this appears to result primarily from indirect transcriptional effects. Lastly, RNase depletion led to the stabilization of many low-abundance potential regulatory RNAs, both in intergenic regions and in the antisense orientation to known transcripts

Ribonuclease Activity of Dis3 Is Required for Mitotic Progression and Provides a Possible Link between Heterochromatin and Kinetochore Function

BACKGROUND: Cellular RNA metabolism has a broad range of functional aspects in cell growth and division, but its role in chromosome segregation during mitosis is only poorly understood. The Dis3 ribonuclease is a key component of the RNA-processing exosome complex. Previous isolation of the dis3-54 cold-sensitive mutant of fission yeast Schizosaccharomyces pombe suggested that Dis3 is also required for correct chromosome segregation. METHODOLOGY/PRINCIPAL FINDINGS: We show here that the progression of mitosis is arrested in dis3-54, and that segregation of the chromosomes is blocked by activation of the mitotic checkpoint control. This block is dependent on the Mad2 checkpoint protein. Double mutant and inhibitor analyses revealed that Dis3 is required for correct kinetochore formation and function, and that this activity is monitored by the Mad2 checkpoint. Dis3 is a member of the highly conserved RNase II family and is known to be an essential subunit of the exosome complex. The dis3-54 mutation was found to alter the RNaseII domain of Dis3, which caused a reduction in ribonuclease activity in vitro. This was associated with loss of silencing of an ura4(+) reporter gene inserted into the outer repeats (otr) and central core (cnt and imr) regions of the centromere. On the other hand, centromeric siRNA maturation and formation of the RITS RNAi effector complex was normal in the dis3-54 mutant. Micrococcal nuclease assay also suggested the overall chromatin structure of the centromere was not affected in dis3-54 mutant. CONCLUSIONS/SIGNIFICANCE: RNase activity of Dis3, a core subunit of exosome, was found to be required for proper kinetochore formation and establishment of kinetochore-microtubule interactions. Moreover, Dis3 was suggested to contribute to kinetochore formation through an involvement in heterochromatic silencing at both outer centromeric repeats and within the central core region. This activity is likely monitored by the mitotic checkpoint, and distinct from that of RNAi-mediated heterochromatin formation directly targeting outer centromeric repeats

The RNA Polymerase Dictates ORF1 Requirement and Timing of LINE and SINE Retrotransposition

Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners—such as the retropseudogenes, SVA, and the SINE, Alu—are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the “pol II Alu transcript” behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing

Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements

A considerable number of genes that code for AU-rich mRNAs including cytokines, growth factors, transcriptional factors, and certain receptors are involved in both chronic inflammation and cancer. Overexpression of these genes is affected by aberrations or by prolonged activation of several signaling pathways. AU-rich elements (ARE) are important cis-acting short sequences in the 3′UTR that mediate recognition of an array of RNA-binding proteins and affect mRNA stability and translation. This review addresses the cellular and molecular mechanisms that are common between inflammation and cancer and that also govern ARE-mediated post-transcriptional control. The first part examines the role of the ARE-genes in inflammation and cancer and sequence characteristics of AU-rich elements. The second part addresses the common signaling pathways in inflammation and cancer that regulate the ARE-mediated pathways and how their deregulations affect ARE-gene regulation and disease outcome

Large-scale profiling of noncoding RNA function in yeast

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs