Eukaryotic non-coding RNAs: new targets for diagnostics and therapeutics?

Non-coding RNAs (ncRNAs) in eukaryotes have recently developed to a very active research area in RNA biology, opening up new strategies for diagnosis and therapies of human disease. Here we introduce and describe the most important classes of eukaryotic ncRNAs: microRNAs (miRNAs), long non-coding RNAs (IncRNAs), and circular RNAs (circRNAs). We further discuss new RNA-based diagnostic and therapeutic concepts.

Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.

Ribonucleoprotein complex formation during pre-mRNA splicing in vitro.

The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

U6 RNA genes from the trypanosomatids Crithidia fasciculata and Leptomonas seymouri have been isolated and sequenced. As in Trypanosoma brucei, the U6 RNA genes in both C. fasciculata and L. seymouri are arranged in close linkage with upstream tRNA genes. The U6 RNA sequences from C. fasciculata and L. seymouri deviate in five and three positions, respectively, from the published T. brucei sequence. Interestingly, both C. fasciculata U6 RNA genes carry a C-->T change at the second position of the ACAGAG hexanucleotide sequence, which is important for splicing function and has been considered phylogenetically invariable. A compensatory base change of the C. fasciculata spliced leader RNA at the highly conserved 5' splice site position +5, G-->A, suggests that an interaction between the 5' splice site region and U6 RNA recently proposed for the yeast cis-splicing system may also occur in trans splicing.

Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.

Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers.

We have developed an in vitro selection procedure that allows the identification and isolation of functional splicing enhancer sequences from any cDNA. It is based on the enhancement of general splicing activity of a pre-mRNA reporter derived from the Drosophila dsx gene. Short DNase I fragments are cloned into a cassette in the second exon of the reporter construct, replacing the natural dsx enhancer. After splicing and reverse transcription-PCR, fragments are recovered from the mRNA product. Applying this selection to the CD44 gene, which undergoes extensive alternative splicing processes, we have identified several novel exonic enhancers. Two of them, which reside in CD44 variable exon 6, were further characterized by mutational analysis and confirmed to function within their natural CD44 context.

Cloning and mutational analysis of the Leptomonas seymouri U5 snRNA gene: function of the Sm site in core RNP formation and nuclear localization.

We have cloned the single-copy gene for the trans -spliceosomal U5 snRNA from the trypanosomatid species Leptomonas seymouri, using U5 RNA affinity selection and cDNA cloning. Sequence comparison revealed that the trans -spliceosomal U5 RNAs from trypanosomatid species share certain characteristic features. Interestingly, the affinity selection procedure yielded-in addition to the bona fide U5 RNA-a closely related small RNA, which can be folded into the same secondary structure, but carries three changes in the loop sequence. This raises the possibility that there may be a larger family of U5-like RNAs in trypanosomes. To study the U5 snRNP assembly and function in trypanosomes we have established a stable expression system in L.seymouri. Two cell lines have been generated that express U5 RNAs with mutations in the Sm site, resulting in a defect of core snRNP formation. In addition, the U5 Sm-mutant RNAs behaved differently in cell fractionation, implying a defect in nuclear localization. In sum, this demonstrates for the first time that the Sm site of trypanosome snRNAs contributes an essential element for stable core RNP assembly and may be important for nuclear localization, in analogy to the Sm site function of cis -spliceosomal snRNAs in higher eucaryotes.

Determinants for cap trimethylation of the U2 small nuclear RNA are not conserved between Trypanosoma brucei and higher eukaryotic organisms.

In most eukaryotic organisms the U2 small nuclear RNA (snRNA) gene is transcribed by RNA polymerase II to generate a primary transcript with a 5' terminal 7-methylguanosine cap structure. Following nuclear export, the U2 snRNA is assembled into a core ribonucleoprotein particle (RNP). This involves binding a set of proteins that are shared by spliceosomal snRNPs to the highly conserved Sm site. Prior to nuclear import, the snRNA-(guanosine-N:2)-methyltransferase appears to interact with the core RNP and hypermethylates the cap structure to 2,2, 7-trimethylguanosine (m(3)G). In the protist parasite Trypanosoma brucei, U-snRNAs are complexed with a set of common proteins that are analogous to eukaryotic Sm antigens but do not have a highly conserved Sm sequence motif, and most U-snRNAs are synthesised by RNA polymerase III. Here, we examined the determinants for m(3)G cap formation in T.brucei by expressing mutant U2 snRNAs in vivo and assaying trimethylation and RNP assembly by immunoprecipitation. Surprisingly, these studies revealed that the Sm-analogous region is not required either for binding of the common proteins or for cap trimethylation. Furthermore, except for the first 24 nt which are part of the U2 promoter, the U2 coding region could be substituted or deleted without affecting cap trimethylation.

Mutational analysis of human U6 RNA: stabilizing the intramolecular helix blocks the spliceosomal assembly pathway.

U6 RNA undergoes several conformational transitions during the spliceosome cycle: after the interaction with U4, the singular form of U6 is converted into the U4-U6 base-paired form, and within the spliceosome, the U4-U6 duplex isomerizes into the active U6-U2 conformation. The secondary structure of the singular form contains an extended 3' stem-loop, the upper part of which (intramolecular helix) most likely reforms in the spliceosome. We have previously shown in the mammalian splicing complementation system that the loop and the three adjacent, highly conserved base pairs of the intramolecular helix function during both the U4-U6 interaction and the first step of splicing. Here we demonstrate that the balanced stability of the lower, less conserved part of the 3' stem-loop is also critical for U4-U6 interaction; however, no specific splicing function could be detected in this region. The analysis of the heterologous interaction between mammalian U4 snRNP and yeast U6 RNA derivatives suggests that there are--in addition to the 3' loop and the stability of the intramolecular helix--specific sequence determinants in the 3' terminal domain of U6 that are important for efficient U4/U6 snRNP assembly.

High CA repeat numbers in intron 13 of the endothelial nitric oxide synthase gene and increased risk of coronary artery disease.

Endothelial nitric oxide synthase (eNOS) plays a key role in vascular homeostasis. Because its product, nitric oxide, possesses vasodilatory and antiatherogenic properties, an altered eNOS function might promote atherosclerosis. We investigated the association between variations in CA repeat copy number [(CA), polymorphism] in intron 13 of the eNOS gene and the risk of coronary artery disease. (CA), polymorphism was investigated in 1000 consecutive patients with angiographically confirmed coronary artery disease and 1000 age- and gender-matched control subjects by a PCR-based fragment length calculation. Twenty-eight different alleles were identified containing 17-44 CA repeats. The presence of one allele containing > or = 38 repeats was associated with an excess risk of coronary artery disease (odds ratio 1.94, 95% confidence interval 1.31-2.86, P = 0.001). Carriers of alleles containing > or = 38 CA repeats were, in particular, overrepresented in the subgroup without common cardiovascular risk factors (odds ratio 3.39, 95% confidence interval 1.30-8.86, P = 0.009). Logistic regression analysis revealed that the (CA), polymorphism proved to be an independent risk factor (relative risk 2.17, 95% confidence interval 1.44-3.27, P = 0.0002). Our findings indicate that high numbers of CA repeats in intron 13 of the eNOS gene are associated with an excess risk of coronary artery disease.

Unexpected diversity in U6 snRNA sequences from trypanosomatids.

We have isolated and sequenced the genes for the trans-spliceosomal U6 small nuclear RNAs (snRNAs) from the trypanosomatid species Leishmania mexicana (Lm) and Phytomonas sp. (Ps). Compared with the Trypanosoma brucei (Tb) U6 snRNA, the Lm U6 snRNA contains only a single additional G-C bp in the 5' terminal stem-loop. In contrast, the Ps U6 snRNA sequence contains a G-->C change at the last nucleotide of the highly conserved and functionally important ACAGAG hexanucleotide and three additional changes in conserved positions. Our results indicate that trans-spliceosomal U6 snRNAs from trypanosomatid species do not always conform to the consensus sequence of cis-spliceosomal U6 snRNAs.

U4 small nuclear RNA genes of trypanosomes: cloning of the Leptomonas seymouri gene and mutational analysis of core snRNP assembly.

Trans mRNA splicing in trypanosomatids requires as cofactors small nuclear RNAs (snRNAs) U2, U4, U5, and U6, in addition to the spliced leader (SL) RNA. To allow a phylogenetic comparison and functional analysis of trypanosomatid U4 snRNAs, we have cloned the single-copy gene for the Leptomonas seymouri U4 snRNA. In addition, a putative U4 snRNA gene from Leishmania tarentolae was identified by database searching. Using an episomal expression system, we introduced mutations into the conserved Sm region of the L. seymouri U4, which is the putative binding site for the common proteins that are present in each of the trans-spliceosomal snRNPs. As demonstrated by CsCl density gradient centrifugation, Sm mutant U4 snRNAs are non-functional in core RNP assembly. Furthermore, we present evidence by cell fractionation that U4 snRNAs with Sm mutations are partially defective in nuclear-cytoplasmic translocation. Taken together this indicates that the Sm site of U4 snRNA is responsible for stable core RNP assembly and nuclear localization.

Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly.

U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.

Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.

Immunological characterization and intracellular localization of trans-spliceosomal small nuclear ribonucleoproteins in Trypanosoma brucei.

Polyclonal antibodies were raised against purified protein components of the U2 small nuclear ribonucleoprotein (snRNP) from Trypanosoma brucei. Through immunoblot and immunoprecipitation analyses three antisera were characterized that reacted specifically with U2 snRNP proteins of molecular weights 40,000 (anti-40K) and 16,500 (anti-16.5K), and with each of four proteins of molecular weights 14,000, 12,500, 10,000, and 8,500 (anti-CP). Anti-40K antibodies specifically immunoprecipitated the U2 snRNP from trypanosomal extracts, whereas anti-CP antibodies recognized several snRNPs, including the SL RNP and the U2 and U4/U6 snRNPs; in addition, minor RNAs were detected, suggesting that a family of snRNPs with common or related protein components exists in trypanosomes. None of these antibodies cross-reacted significantly with total mammalian snRNP proteins, indicating that the trypanosomal snRNP proteins are immunologically distinct from their mammalian counterparts. Using immunofluorescence microscopy, the snRNP proteins exhibited a differential cellular distribution. Whereas the 40-kDa protein is localized exclusively in the nucleus, with the nucleolus being excluded, a fraction of the common proteins also resides in the cytoplasm.