Suppression of mutant Kirsten-RAS (KRAS)-driven pancreatic carcinogenesis by dual-specificity MAP kinase phosphatases 5 and 6.

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRAS-driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRAS-driven pancreatic hyperplasia.

Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines.

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins.

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis.

FMN2 is a novel regulator of the cyclin-dependent kinase inhibitor p21.

We have identified the human FMN2 gene as a novel target regulated by induction of p14ARF and by multiple other stress responses, including DNA damage and hypoxia, which have in common activation of cell cycle arrest. We showed that increased expression of the FMN2 gene following p14ARF induction is caused, at the transcriptional level, by relief of repression by RelA and E2F1, which, under non-induced conditions, bind the FMN2 promoter. Increased FMN2 protein levels promote cell cycle arrest by inhibiting the degradation of p21, and our data show that control of p21 stability is a key part of the mechanism that regulates p21 induction.

Analysis of human protein replacement stable cell lines established using snoMEN-PR vector.

The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN) vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP)-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs.

Identification and functional characterization of FMN2, a regulator of the cyclin-dependent kinase inhibitor p21.

The ARF tumor suppressor is a central component of the cellular defense against oncogene activation in mammals. p14ARF activates p53 by binding and inhibiting HDM2, resulting, inter alia, in increased transcription and expression of the cyclin-dependent kinase inhibitor p21 and consequent cell-cycle arrest. We analyzed the effect of p14ARF induction on nucleolar protein dynamics using SILAC mass spectrometry and have identified the human Formin-2 (FMN2) protein as a component of the p14ARF tumor suppressor pathway.

Human box C/D snoRNA processing conservation across multiple cell types.

Small nucleolar RNAs (snoRNAs) function mainly as guides for the post-transcriptional modification of ribosomal RNAs (rRNAs). In recent years, several studies have identified a wealth of small fragments (<35 nt) derived from snoRNAs (termed sdRNAs) that stably accumulate in the cell, some of which may regulate splicing or translation. A comparison of human small RNA deep sequencing data sets reveals that box C/D sdRNA accumulation patterns are conserved across multiple cell types although the ratio of the abundance of different sdRNAs from a given snoRNA varies.

From snoRNA to miRNA: Dual function regulatory non-coding RNAs.

Small nucleolar RNAs (snoRNAs) are an ancient class of small non-coding RNAs present in all eukaryotes and a subset of archaea that carry out a fundamental role in the modification and processing of ribosomal RNA. In recent years, however, a large proportion of snoRNAs have been found to be further processed into smaller molecules, some of which display different functionality. In parallel, several studies have uncovered extensive similarities between snoRNAs and other types of small non-coding RNAs, and in particular microRNAs.

Identification of human miRNA precursors that resemble box C/D snoRNAs.

There are two main classes of small nucleolar RNAs (snoRNAs): the box C/D snoRNAs and the box H/ACA snoRNAs that function as guide RNAs to direct sequence-specific modification of rRNA precursors and other nucleolar RNA targets. A previous computational and biochemical analysis revealed a possible evolutionary relationship between miRNA precursors and some box H/ACA snoRNAs. Here, we investigate a similar evolutionary relationship between a subset of miRNA precursors and box C/D snoRNAs.

Analysis of human small nucleolar RNAs (snoRNA) and the development of snoRNA modulator of gene expression vectors.

Human small nucleolar RNAs (snoRNAs) that copurify with nucleoli isolated from HeLa cells have been characterized. Novel fibrillarin-associated snoRNAs were detected that allowed the creation of a new vector system for the targeted knockdown of one or more genes in mammalian cells. The snoMEN (snoRNA modulator of gene expressioN) vector technology is based on snoRNA HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets.

Human miRNA precursors with box H/ACA snoRNA features.

MicroRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) are two classes of small non-coding regulatory RNAs, which have been much investigated in recent years. While their respective functions in the cell are distinct, they share interesting genomic similarities, and recent sequencing projects have identified processed forms of snoRNAs that resemble miRNAs. Here, we investigate a possible evolutionary relationship between miRNAs and box H/ACA snoRNAs.

HNF-1 regulates the promoter activity of the HP-27 gene.

The hibernation-specific HP-27 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. In the promoter region, the chipmunk gene has a potential HNF-1 binding site, and the tree squirrel gene has two base substitutions in the corresponding sequence. In this paper, we investigated the role of HNF-1 in the HP-27 gene promoter activity.

Comparative study of HP-27 gene promoter activities between the chipmunk and tree squirrel.

The chipmunk hibernation-specific protein HP-27 is a component of the 140-kDa complex that decreases in the blood during hibernation. Although the HP-27 gene is detected in both the chipmunk, a hibernating species of the squirrel family, and the tree squirrel, a nonhibernating species, it is expressed only in the chipmunk, in a liver-specific manner. To understand the difference in HP-27 gene expression between the chipmunk and tree squirrel, we isolated chipmunk and tree squirrel HP-27 genomic clones, and compared their promoter activities.