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.

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.

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.

The spliceosomal snRNP core complex of Trypanosoma brucei: cloning and functional analysis reveals seven Sm protein constituents.

Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4/U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.

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.

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.

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.

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Domain analysis of human U5 RNA. Cap trimethylation, protein binding, and spliceosome assembly.

We have analyzed the sequence requirements of the human U5 RNA during small nuclear ribonucleoprotein (snRNP) and spliceosome assembly. A collection of mutant derivatives of the human U5 RNA gene was constructed in a U1 expression vector and transiently transfected in mammalian cells. Using immunoprecipitation and affinity selection assays, the cap trimethylation, the binding of Sm proteins and of the U5 snRNP-specific protein p220, as well as the assembly of the U4/U5/U6 triple snRNP and of spliceosomes were determined. By mutational analysis we were able to assign distinct functions to several structural elements of the human U5 RNA. Efficient binding of the Sm proteins requires the 3' stem-loop. Both the Sm protein-binding site and the 3' stem-loop are necessary for the formation of the trimethyl guanosine cap, consistent with Sm protein binding being a prerequisite for cap trimethylation. Specific elements of the U5 RNA 5' stem-loop contribute to efficient p220 association, in particular stem Ib. Interestingly, the highly conserved loop I appears to be a multifunctional element; in addition to its function in splice-site selection the 5' loop is involved in binding of p220 and in the assembly of the U4/U5/U6 triple snRNP. In sum, this mutational analysis has identified four functional domains of the human U5 RNA.

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.

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.

Spliced leader-associated RNA of trypanosomes. Sequence conservation and association with protein components common to trans-spliceosomal ribonucleoproteins.

A spliced leader-associated (SLA) RNA recently identified in Trypanosoma brucei has been proposed as a U5 RNA analog in trans-splicing, based on in vivo psoralen cross-linking to the spliced leader (SL) RNA and a short stretch of homology to a conserved U5 RNA sequence. Here we show that the SLA RNA is in the form of an RNP containing common proteins that also occur in the known trans-spliceosomal snRNPs of T. brucei, the SL, U2, and U4/U6 snRNPs. Therefore the SLA RNP is a member of a larger family of related RNPs. The immunoprecipitation of the SLA RNA by antibodies against common proteins does not require an association between the SLA RNA and the SL RNP. To identify sequences of potential functional importance and to establish a secondary structure model, we have sequenced the SLA RNA and cloned its gene from a related trypanosomatid, Leptomonas seymouri. A sequence comparison between the T. brucei and L. seymouri SLA RNAs reveals that the U5-homologous sequence element is not conserved.

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.

Splicing function of mammalian U6 small nuclear RNA: conserved positions in central domain and helix I are essential during the first and second step of pre-mRNA splicing.

On the basis of mutational analyses in yeast, the highly conserved ACAGAGA sequence of U6 small nuclear RNA (snRNA) and the adjacent U6-U2 helix I have been proposed to be part of the active center of the spliceosome. We report here a detailed analysis of the human U6 snRNA sequence requirements during the first and second step of splicing, using a mammalian in vitro splicing-complementation system and a mutational approach. Positions A53G54C55 (helix Ib) were identified as important specifically for the first step, but not for spliceosome assembly. A45 of the ACAGAGA sequence and U52 of helix Ia function during the second step; in addition, the bulge separating helices Ia and Ib appears critical for the second step. In contrast, no splicing-essential sequences could be identified in the central domain upstream of the ACAGAGA sequence. In sum, our data demonstrate for the mammalian splicing system that discrete positions within the ACAGAGA sequence and helix I of U6 snRNA function during the first and second step of splicing, suggesting that these two sequence elements are closely associated with the catalytic center of the spliceosome. Comparison with previous results in yeast indicates a fundamental conservation of the U6 snRNA function in the pre-mRNA splicing mechanism.

Conformational changes of U6 RNA during the spliceosome cycle: an intramolecular helix is essential both for initiating the U4-U6 interaction and for the first step of slicing.

During each spliceosome cycle, U6 RNA undergoes several conformational changes, involving the formation and disruption of base-pairing interactions with U4 and U2 RNAs. By use of a mutational approach we have focused on the stem II region of U6, which can adopt alternative conformations: In the singular form of U6, it can form an intramolecular stem-loop structure; in the U4/U6 snRNP, the stem II region base-pairs with U4 RNA; in the active spliceosome, this region has been proposed to fold back into an intramolecular U6 helix in the context of a U6-U2 structure. Using chemical modification/interference assays and a mutational approach we found that the 3' terminal loop of the singular U6 (nucleotides 65-69) is essential for initiating the U4-U6 base-pairing interaction. A series of point mutations in the adjacent helix was designed to alter the stability of the intramolecular helix. Stabilizing mutations inhibited the formation of the U4/U6 snRNP. In contrast, mutant U6 RNAs with a destabilized intramolecular helix were still active in U4-U6 interaction and spliceosome assembly; however, their ability to support the first step of splicing was strongly reduced, suggesting that the intramolecular U6 helix has an important function in the first step of splicing. Affinity-purified U4 snRNP and U6 RNA did not assemble into a stable U4/U6 snRNP, unless complemented by nuclear extract, indicating that a protein factor (or factors) is necessary for the U4-U6 interaction. In sum, these data demonstrate that the stem II region of U6 functions both in U4-U6 interaction and in the first step of splicing; they also provide evidence that the balanced stability of different conformations of U6 RNA is critical for its function.

Assembly of the U2 small nuclear ribonucleoprotein from Trypanosoma brucei. A mutational analysis.

trans-Splicing in trypanosomes requires the functions of U2 and U4/U6 small nuclear (sn) RNPs. We have analyzed protein binding and assembly of the Trypanosoma brucei U2 snRNP, using specific antibodies against U2 snRNP proteins and in vitro reconstitution assays of U2 deletion derivatives and human-trypanosome hybrid RNAs. Stable binding of both the U2-specific 40-kDa and the common proteins requires only the 3'-terminal domain (stem-loop IIb, single-stranded region, and stem-loop IV), with loop IV providing the critical sequence determinant; stem-loop IV suffices for binding of the 40 kDa-protein, but not of the common proteins; surprisingly, the sequence of the "Sm-analogous" single-stranded region between stem-loops IIb and IV is not essential for protein binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the assembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP assembly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from that of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.

The trans-spliceosomal U2 snRNP protein 40K of Trypanosoma brucei: cloning and analysis of functional domains reveals homology to a mammalian snRNP protein.

Through immunoscreening we have isolated a cDNA encoding the trans-spliceosomal U2 snRNP-specific 40 kDa protein of Trypanosoma brucei. The protein has a predicted molecular weight of 36.6 kDa and shows 31% amino acid identity with the human U2 snRNP A' protein of 28.4 kDa. The homology between the trypanosome and human protein sequences is restricted to the N-terminal half where they share a series of six leucine repeat motifs. Sequence alignment revealed three 40K-specific regions: a C-terminal extension and two insertions, one of which makes up a seventh leucine repeat. Bacterially expressed 40K protein efficiently bound RNA by itself in a nonspecific manner; this general RNA binding activity was located to a region in the C-terminal half overlapping with the leucine repeat domain. U2 RNA-specific interaction required the presence of other trypanosome proteins and depended upon the loop IV sequence of U2 RNA. Deletion analysis of the 40K protein demonstrated the leucine repeats, including the 40K-specific, seventh repeat, to be essential for specific U2 RNP assembly, most likely through their role as an interface for protein-protein interaction.