SUMMARY Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.

Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.

DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.

Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is wellrepresented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in AGI at 37°C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5°C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G-U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.Stabilities of RNA duplexes and secondary structures of RNAs are often predicted by using free-energy parameters from a nearest-neighbor model (1-5). Sometimes, however, predictions are inconsistent with experimental data (6-10). One factor hindering successful predictions is that the reliability of parameters was limited by the availability of model oligonucleotides (2). Recent breakthroughs in synthesis of RNA oligoribonucleotides (11-16) permit design of oligonucleotides to provide improved parameters. This paper presents thermodynamic parameters derived from data on 45 complementary RNA duplexes. The parameters are able to predict the stabilities of RNA duplexes within the limits ofthe nearest-neighbor model. MATERIALS AND METHODSChoice of Sequences. Sequences were selected to minimize errors in the free-energy change for duplex formation at 37°C, AG97 (17,18). Thus, as much as possible, melting temperatures at 0.1 mM are near 37°C to minimize extrapolation. The oligomers were also chosen to independently represent all 10 nearest-neighbor sequences comprising Watson-Crick base pairs.Oligonucleotide Synthesis. Oligonucleotides not reported elsewhere were synthesized on solid support using phosphoramidite procedures and purified as described (11,19). Purities were confirmed by high-performance liquid chromatography for all oligomers.Thermodynamic Parameters. Absorbance vs. temperature melting curves were measured in 1 M NaCl/0.005 M Na2HPO4/0.5 mM EDTA (disodium salt), pH 7, as described (11). Concentrations were determined from the high-temperature absorbance using extinction coefficients calculated as described (20). In units of 0.1 mM-1 cm-1, calculated hightemperature extinction coefficients at 280 nm not reported elsewhere are as follows: GUGCAC, 2.77; GUCUAGAC, 3.66; GAUAUAUC, 3.05; GUAUAUAC, 3.00. Thermodynamic parameters of helix formation were obtained by two methods. (t) Individual melting curves were fit to a two-state model with sloping baselines and the enthalpy and entropy changes derived from the fits were averaged (21), and (ii) reciprocal melting temperature, tm-1, vs. log (CT) was plot...

MicroRNAs (miRNAs) are endogenously expressed 20 -24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.The first miRNA 1 was identified in Caenorhabditis elegans as a gene important for timing of larval development (5). miRNAs have since been implicated in many processes in invertebrates, including cell proliferation and apoptosis (6, 7), fat metabolism (6), and neuronal patterning (8). As many miRNAs are conserved across species (9 -11), they are likely to be involved in developmental processes in all animals. Only a few mammalian miRNAs have been assigned any function, and at least two of these are involved in developmental processes: miR-181 promotes B cell development in mice (12) and miR196a regulates several Hox genes (13), which code for a family of transcription factors involved in various developmental programs in animals (14).We hypothesized that miRNAs may play a role in maturation of human adipocytes. Understanding the molecular events involved in adipocyte differentiation is of interest for development of therapeutics for metabolic diseases such as obesity and diabetes. In vitro cell culture systems, such as human primary subcutaneous pre-adipocytes, have been crucial in uncovering signaling pathways important for adipocyte differentiation (15). These cells can be cultured with differentiation-promoting hormonal stimuli, causing them to develop into cells that morphologically and functionally resemble mature adipocytes. In this study we have inhibited a panel of miRNAs in pre-adipocytes using antisense oligonucleotides and evaluated the effect on adipocyte differentiation. Combined with expression analysis of miRNAs in differentiating adipocytes by microarray, one miRNA, miR-143, was identified which normally promotes adipocyte differentiation. These results indicate that miRNAs do play a role in adipocyte differentiation and are potential therapeutic targets for obesity and metabolic diseases. EXPERIMENTAL PROCEDURESOligonucleotide Synthesis-Oligonucleotides were prepared using conventional phosphoramidite chemistry and...

Multiple mechanisms have evolved to regulate the eukaryotic genome. We have identified CTN-RNA, a mouse tissue-specific approximately 8 kb nuclear-retained poly(A)+ RNA that regulates the level of its protein-coding partner. CTN-RNA is transcribed from the protein-coding mouse cationic amino acid transporter 2 (mCAT2) gene through alternative promoter and poly(A) site usage. CTN-RNA is diffusely distributed in nuclei and is also localized to paraspeckles. The 3'UTR of CTN-RNA contains elements for adenosine-to-inosine editing, involved in its nuclear retention. Interestingly, knockdown of CTN-RNA also downregulates mCAT2 mRNA. Under stress, CTN-RNA is posttranscriptionally cleaved to produce protein-coding mCAT2 mRNA. Our findings reveal a role of the cell nucleus in harboring RNA molecules that are not immediately needed to produce proteins but whose cytoplasmic presence is rapidly required upon physiologic stress. This mechanism of action highlights an important paradigm for the role of a nuclear-retained stable RNA transcript in regulating gene expression.

The formation of mature mRNAs in vertebrates involves the cleavage and polyadenylation of the pre-mRNA, 10-30 nt downstream of an AAUAAA or AUUAAA signal sequence. The extensive cDNA data now available shows that these hexamers are not strictly conserved. In order to identify variant polyadenylation signals on a large scale, we compared over 8700 human 3Ј untranslated sequences to 157,775 polyadenylated expressed sequence tags (ESTs), used as markers of actual mRNA 3Ј ends. About 5600 EST-supported putative mRNA 3Ј ends were collected and analyzed for significant hexameric sequences. Known polyadenylation signals were found in only 73% of the 3Ј fragments. Ten single-base variants of the AAUAAA sequence were identified with a highly significant occurrence rate, potentially representing 14.9% of the actual polyadenylation signals. Of the mRNAs, 28.6% displayed two or more polyadenylation sites. In these mRNAs, the poly(A) sites proximal to the coding sequence tend to use variant signals more often, while the 3Ј-most site tends to use a canonical signal. The average number of ESTs associated with each signal type suggests that variant signals (including the common AUUAAA) are processed less efficiently than the canonical signal and could therefore be selected for regulatory purposes. However, the position of the site in the untranslated region may also play a role in polyadenylation rate.The 3Ј untranslated regions (UTRs) of eukaryotic mRNAs contain regulatory elements affecting mRNA translation, stability, and transport. Mature 3Ј UTRs are formed by polyadenylation of the pre-mRNA, a coupled reaction involving endonucleolytic cleavage followed by poly(A) synthesis. A significant fraction of mRNAs display multiple polyadenylation sites (Gautheret et al. 1998). The choice of poly(A) sites may influence the stability, translation efficiency, or localization of an mRNA in a tissue-or disease-specific manner (Edwalds-Gilbert et al. 1997). In the mammalian system, effective polyadenylation requires two main sequence components: a highly conserved AAUAAA signal located 10-30 nucleotide 5Ј to the cleavage site and a more variable GU-rich element, 20-40 bases 3Ј of the site (see Proudfoot 1991; Colgan and Manley 1997 for reviews). Although the AAUAAA signal is often considered to be present in 90% of the mRNAs and replaced by a AUUAAA variant in the other 10% (Wahle and Keller 1996; Colgan and Manley 1997), alternate signals are certainly present in a significant fraction of the 3Ј ends (Claverie 1997;Gautheret et al. 1998;Tabaska and Zhang 1999;Graber et al. 1999).The expressed sequence tag (EST) database, dbEST (Boguski et al. 1993), which contains highly redundant partial cDNAs, especially from the 3Ј UTRs, is a rich source of information on mRNA 3Ј ends. Analyzing clustered EST sequences, we previously identified multiple cases of alternate polyadenylation in mRNA (Gautheret et al. 1998). Based on a public EST collection now containing over 1.4 million human sequences, the present work focuses on the region immediatel...

MALAT1 is a long non-coding RNA whose expression level was originally identified as a predictor of metastasis of non-small cell lung tumors and was subsequently shown to be over-expressed in many human cancers. Here, we have identified a highly conserved tRNA-like small RNA of 61 nucleotides that originates from the MALAT1 locus and is broadly expressed in human tissues. In contrast to the long MALAT1 transcript that localizes to nuclear speckles, the small RNA exclusively localizes to the cytoplasm. We show that RNase P cleaves downstream of a genomically encoded poly(A)-rich tract to simultaneously generate the 3’ end of the abundant MALAT1 transcript and the 5’ end of the small RNA. Our findings reveal a novel 3’ end processing mechanism by which a single locus can yield both a stable nuclear retained non-coding RNA with a short poly(A) tail-like moiety and a small tRNA-like cytoplasmic RNA.