Abundant Small RNAs Research Articles

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ((1)O(2)) in Rhodobacter sphaeroides. Our study reports the genome-wide search for small RNAs (sRNAs) involved in the regulatory response to (1)O(2). By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with (1)O(2) or superoxide (O(-)(2)). One sRNA was specifically induced by (1)O(2) and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by (1)O(2) and O(-)(2) were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH(I) and RpoH(II). The most abundant sRNA was processed in the presence of (1)O(2) but not by O(-)(2). From this and a second sRNA a conserved 3'-segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half-lives, abundance and processing of (1)O(2)-affected sRNAs. Orthologues of three sRNA genes are present in different alpha-proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by (1)O(2) exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs

Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNA(Gln) is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro.

Characterization and comparative profiling of the small RNA transcriptomes in two phases of locust

High-throughput sequencing of the small RNA transcriptome of locust reveals differences in post-transcriptional regulation between solitary and swarming phases and provides insights into the evolution of insect small RNAs.

Criteria for Annotation of Plant MicroRNAs

MicroRNAs (miRNAs) are approximately 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

Stress-induced tRNA-derived RNAs: a novel class of small RNAs in the primitive eukaryote Giardia lamblia

Giardia lamblia is an early diverging and evolutionarily successful protozoan as it can enter into a dormant cyst stage from a vegetative trophozoite. During dormant stage, its metabolic rate decreases dramatically. However, to date, the regulatory molecules participating in the initiation and maintenance of this process have not been fully investigated. In this study, we have identified a class of abundant small RNAs named sitRNAs, which are ∼46 nucleotides in length and accumulate in G. lamblia encysting cultures. Remarkably, they are derived from the 3′ portion of fully matured tRNAs by cleavage of the anticodon left arm, with the 3′ terminal CCA triplex still connected. During differentiation, only a limited portion of mature tRNAs is cleaved, but this cleavage occurs almost in the entire tRNA family. sitRNAs begin to accumulate as early as 3 h after initiation of encystation and are maintained at a relatively stable level during the whole process, exhibiting an expression peak at around 24 hr. Our studies further show that sitRNAs can be induced by several other stress factors, and in the case of serum deprivation, both tRNAs and sitRNAs degrade rapidly, with the accumulation of tRNA being halved. Our results may provide new insight into a novel mechanism for stressed G. lamblia to regulate gene expression globally.

Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma

AbstractCyclin D1 (CCND1) is a well-known regulator of cell-cycle progression. It is overexpressed in several types of cancer including breast, lung, squamous, neuroblastoma, and lymphomas. The most well-known mechanism of overexpression is the t(11;14)(q13;q32) translocation found in mantle cell lymphoma (MCL). It has previously been shown that truncated CCND1 mRNA in MCL correlates with poor prognosis. We hypothesized that truncations of the CCND1 mRNA alter its ability to be down-regulated by microRNAs in MCL. MicroRNAs are a new class of abundant small RNAs that play important regulatory roles at the posttranscriptional level by binding to the 3′ untranslated region (UTR) of mRNAs blocking either their translation or initiating their degradation. In this study, we have identified the truncation in CCND1 mRNA in MCL cell lines. We also found that truncated CCND1 mRNA leads to increased CCND1 protein expression and increased S-phase cell fraction. Furthermore, we demonstrated that this truncation alters miR-16-1 binding sites, and through the use of reporter constructs, we were able to show that miR-16-1 regulates CCND1 mRNA expression. This study introduces the role of miR-16-1 in the regulation of CCND1 in MCL.

Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes

Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.

Truncations in CCND1 mRNA 3′ UTR Alters microRNA Regulation in Mantle Cell Lymphoma.

Mantle Cell Lymphoma (MCL) represents only 5–10% of all non-Hodgkins lymphomas, making it an uncommon but difficult form of lymphoma to treat. It has a poor prognosis among the B cell lymphomas with median survival of three years. The genetic hallmark of MCL is the t(11,14) translocation causing amplification of cyclin D1 (CCND1), a known cell cycle regulator which is overexpressed in many other cancers. MicroRNAs (miRNA) are a new class of abundant small RNAs that play important regulatory roles at the post transcriptional level. They act by binding to the 3′ untranslated region (UTR) of mRNAs and block either their translation or initiate their degradation. Recent reports have shown truncations in the CCND1 3′ UTR occur in MCL and indicate a worse prognosis. We hypothesized that truncations in 3′ UTR of CCND1 alter it's regulation by microRNAs. Based on bioinformatics, we identified microRNA 16 with putative docking sites in the 3′UTR of CCND1. Mir-16 has been implicated as a cell cycle regulator. We identified 2 cell lines (Jeko-1 and Z138) with truncations in CCND1 3′ UTR and demonstrated increased CCND1 mRNA expression by qRT-PCR, increased protein expression by western blot, and higher proliferative potential by cell cycle. We prepared a reporter construct by ligating the full length 3′ UTR of CCND1 to GFP. We then co-transfected this construct with mimics of mir-16 into a cancer cell line and demonstrated downregulation of CCND1 protein expression by flow cytometry. In the MCL cell line Granta-519 with non-truncated CCND1, transfection with mimics of mir-16 deminstrated decreased expression of CCND1 mRNA. These studies suggest that the overexpression of CCDN1 In MCL may result from altered regulation of gene expression from loss of a miRNA regulatory site and may give new clues into the patho-biology of this disease and insights into possible new therapies.

MicroRNA regulation in mantle cell lymphoma

8056 Mantle Cell Lymphoma (MCL) represents only 5–10% of all non-Hodgkins lymphomas, making it an uncommon but difficult form of lymphoma to treat. It has the worst prognosis among the B cell lymphomas with median survival of three years. The genetic hallmark of MCL is a t(11;14) q32 translocation which results in ectopic and dysregulated expression of cyclin D1. Recent comparative genomic hybridization studies have identied Syk as another gene important in the pathogenesis of MCL. Previous studies have suggested that post transcriptional regulation of Cyclin D1 and Syk may be important the pathogenesis of in MCL. MicroRNAs are a new class of abundant small RNAs that play important regulatory roles at the post transcriptional level by binding to the 3’ untranslated region (UTR) of mRNAs blocking either their translation or initiating their degradation. There have been numerous reports of misregulation of microRNAs and their targets in human cancers. We hypothesized that altered microRNA regulation of cyclin D1 and/or Syk may be present in MCL. Based on bioinformatics, we identified 8 microRNAs and their putative docking sites in either Cyclin D1 and Syk. We then examined their integrity in MCL cell lines, and identified a mutation in the 3’UTR of Syk at the docking site of 1 mir-452* and a SNP in mir-458. Using a GFP reporter construct with the mutated Syk 3’UTR we demonstrated that this mutation resulted in altered microRNA function. We also show that mimics of the microRNA leads to down regulation of Syk protein. This data suggests that microRNA regulation of important genes in MCL may be compromised and play a role in the development and progression of this disease. No significant financial relationships to disclose.

The Nuclear Pore Protein AtTPR Is Required for RNA Homeostasis, Flowering Time, and Auxin Signaling

Nuclear pore complexes (NPCs) mediate the transport of RNA and other cargo between the nucleus and the cytoplasm. In vertebrates, the NPC protein TRANSLOCATED PROMOTER REGION (TPR) is associated with the inner filaments of the nuclear basket and is thought to serve as a scaffold for the assembly of transport machinery. In a screen for mutants that suppress the expression of the floral inhibitor FLOWERING LOCUS C, we identified lesions in the Arabidopsis (Arabidopsis thaliana) homolog of TPR (AtTPR). attpr mutants exhibit early-flowering and other pleiotropic phenotypes. A possible explanation for these developmental defects is that attpr mutants exhibit an approximately 8-fold increase in nuclear polyA RNA. Thus AtTPR is required for the efficient export of RNA from the nucleus. Microarray analysis shows that, in wild type, transcript abundance in the nuclear and total RNA pools are highly correlated; whereas, in attpr mutants, a significantly larger fraction of transcripts is enriched in either the nuclear or total pool. Thus AtTPR is required for homeostasis between nuclear and cytoplasmic RNA. We also show that the effects of AtTPR on small RNA abundance and auxin signaling are similar to that of two other NPC-associated proteins, HASTY (HST) and SUPPRESSOR OF AUXIN RESISTANCE3 (SAR3). This suggests that AtTPR, HST, and SAR3 may play related roles in the function of the nuclear pore.

Micromanaging the Immune System

MicroRNAs (miRNAs) are abundant small RNA species that have emerged as key regulators in many biological processes. Rodriguez et al . observed that mice deficient in miRNA-155 develop spontaneous inflammation of the lungs and have accompanying defects in antigen presentation as well as in T cell and B cell function. Exploring the same miRNA, Thai et al . observed a similar T and B cell deficiency that resulted in a suboptimal response of the germinal center, which is needed for T cell-mediated antibody production. Although both studies provide some evidence for how this miRNA mediates its effects, the next important step will be to identify the precise mechanism and critical target genes involved. A. Rodriguez, E. Vigorito, S. Clare, M. V. Warren, P. Couttet, D. R. Soond, S. van Dongen, R. J. Grocock, P. P. Das, E. A. Miska, D. Vetrie, K. Okkenhaug, A. J. Enright, G. Dougan, M. Turner, A. Bradley, Requirement of bic/microRNA-155 for normal immune function. Science 316 , 608-611 (2007). [Abstract] [Full Text] T.-H. Thai, D. P. Calado, S. Casola, K. M. Ansel, C. Xiao, Y. Xue, A. Murphy, D. Frendewey, D. Valenzuela, J. L. Kutok, M. Schmidt-Supprian, N. Rajewsky, G. Yancopoulos, A. Rao, K. Rajewsky, Regulation of the germinal center response by microRNA-155. Science 316 , 604-608 (2007). [Abstract] [Full Text]

Small RNAs hit the big time

Small RNAs hit the big time

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.

6S RNA Regulation of pspF Transcription Leads to Altered Cell Survival at High pH

6S RNA is a highly abundant small RNA that regulates transcription through direct interaction with RNA polymerase. Here we show that 6S RNA directly inhibits transcription of pspF, which subsequently leads to inhibition of pspABCDE and pspG expression. Cells without 6S RNA are able to survive at elevated pH better than wild-type cells due to loss of 6S RNA-regulation of pspF. This 6S RNA-dependent phenotype is eliminated in pspF-null cells, indicating that 6S RNA effects are conferred through PspF. Similar growth phenotypes are seen when PspF levels are increased in a 6S RNA-independent manner, signifying that changes to pspF expression are sufficient. Changes in survival at elevated pH most likely result from altered expression of pspABCDE and/or pspG, both of which require PspF for transcription and are indirectly regulated by 6S RNA. 6S RNA provides another layer of regulation in response to high pH during stationary phase. We propose that the normal role of 6S RNA at elevated pH is to limit the extent of the psp response under conditions of nutrient deprivation, perhaps facilitating appropriate allocation of diminishing resources.

How to find small non-coding RNAs in bacteria

Small non-coding RNAs (sRNAs) have attracted considerable attention as an emerging class of gene expression regulators. In bacteria, a few regulatory RNA molecules have long been known, but the extent of their role in the cell was not fully appreciated until the recent discovery of hundreds of potential sRNA genes in the bacterium Escherichia coli. Orthologs of these E. coli sRNA genes, as well as unrelated sRNAs, were also found in other bacteria. Here we review the disparate experimental approaches used over the years to identify sRNA molecules and their genes in prokaryotes. These include genome-wide searches based on the biocomputational prediction of non-coding RNA genes, global detection of non-coding transcripts using microarrays, and shotgun cloning of small RNAs (RNomics). Other sRNAs were found by either co-purification with RNA-binding proteins, such as Hfq or CsrA/RsmA, or classical cloning of abundant small RNAs after size fractionation in polyacrylamide gels. In addition, bacterial genetics offers powerful tools that aid in the search for sRNAs that may play a critical role in the regulatory circuit of interest, for example, the response to stress or the adaptation to a change in nutrient availability. Many of the techniques discussed here have also been successfully applied to the discovery of eukaryotic and archaeal sRNAs.

Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

Abundant approximately 28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (DeltaDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These DeltaDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation DeltaDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, DeltaDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

Small regulatory RNAs in mammals

Mammalian cells harbor numerous small non-protein-coding RNAs, including small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability, translation, and quite possibly transcription and splicing. These RNAs are processed by multistep pathways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal stem cells and the brain, and some are imprinted. Small RNAs control a wide range of developmental and physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The extent of transcription of non-coding sequences and the abundance of small RNAs suggests the existence of an extensive regulatory network on the basis of RNA signaling which may underpin the development and much of the phenotypic variation in mammals and other complex organisms and which may have different genetic signatures from sequences encoding proteins.

Activation of p53 or Loss of the Cockayne Syndrome Group B Repair Protein Causes Metaphase Fragility of Human U1, U2, and 5S Genes

Infection by adenovirus 12, transfection with the Ad12 E1B 55 kDa gene, or activation of p53 cause metaphase fragility of four loci ( RNU1, PSU1, RNU2, and RN5S) each containing tandemly repeated genes for an abundant small RNA (U1, U2, and 5S RNA). We now show that loss of the Cockayne syndrome group B protein (CSB) or overexpression of the p53 carboxy-terminal domain induces fragility of the same loci; moreover, p53 interacts with CSB in vivo and in vitro. We propose that CSB functions as an elongation factor for transcription of structured RNAs, including some mRNAs. Activation of p53 would inhibit CSB, stalling transcription complexes and locally blocking chromatin condensation. Impaired transcription elongation may also explain the diverse clinical features of Cockayne syndrome.

The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.

Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene.

EXPRESSION OF LATENT HHV-8 GENES

39 Most spindle cells in a KS lesion display evidence of latent HHV-8 infection. To identify the viral genes expressed in latency we have employed several expression-based stragegies. Our earliest cDNA expression strategy yielded T0.7, a highly abundant small RNA widely expressed in KS lesions. Recently we have found that HHV8-infected B lymphoma cells contain variants of this transcript that are substantially longer than that initially described, and variable from tumor to tumor. Some of this variation is due to variations from isolate to isolate in the structure of genomic DNA in this region, and some to novel promoter usage. The new promoters described here are active in B, endothelial and epithelial cells, and their RNA products encode novel proteins whose structures differ greatly from that predicted to be generated from T0.7. Adjacent to this region is a cluster of latent genes identified by screening the HHV8 genome for sequences that are strongly expressed in BCBL-1 cells prior to induction with TPA. These include orfs 71,72 and 73. The fine structures of the transcripts spanning this region have been determined by Northern blotting, RNase mapping, primer extension and RACE procedures. Orf 73/LANA transcripts are polycistronic and exist in both spliced and unspliced forms. Orf 72/v-cyc transcripts are bicistronic and some may initiate just 5′ to the orf. Interestingly, no monocistronic orf 71 mRNAs have yet been identified. Sequences 5′ to orfs 73 and 72 are active in promoting expression of promoterless luciferase genes, suggesting that they contain functional latency promoters. The structure and regulation of these promoters is under study.