The removal of non-coding sequences, introns, from the mRNA precursors is an essential step in eukaryotic gene expression. U12-type introns are a minor subgroup of introns, distinct from the major or U2-type introns. U12-type introns are present in most eukaryotes but only account for less than 0.5% of all introns in any given genome. They are processed by a specific U12-dependent spliceosome, which is similar to, but distinct from, the major spliceosome. U12-type introns are spliced somewhat less efficiently than the major introns, and it is believed that this limits the expression of the genes containing such introns. Recent findings on the role of U12-dependent splicing in development and human disease have shown that it can also affect multiple cellular processes not directly related to the functions of the host genes of U12-type introns. At the same time, advances in understanding the regulation and phylogenetic distribution of the minor spliceosome are starting to shed light on how the U12-type introns and the minor spliceosome may have evolved. © 2012 John Wiley & Sons, Ltd.
Alternative pre-mRNA splicing is typically regulated by specific protein factors that recognize unique sequence elements in pre-mRNA and affect, directly or indirectly, nearby splice site usage. We show that 5' splice site sequences (5'ss) of U12-type introns, when repeated in tandem, form a U11 snRNP-binding splicing enhancer, USSE. Binding of U11 to the USSE regulates alternative splicing of U2-type introns by activating an upstream 3'ss. The U12-type 5'ss-like sequences within the USSE have a regulatory role and do not function as splicing donors. USSEs, present both in animal and plant genes encoding the U11/U12 di-snRNP-specific 48K and 65K proteins, create sensitive switches that respond to intracellular levels of functional U11 snRNP and alter the stability of 48K and 65K mRNAs. We conclude that U11 functions not only in 5'ss recognition in constitutive splicing, but also as an activator of U2-dependent alternative splicing and as a regulator of the U12-dependent spliceosome.
Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2a rmc1 mutants resulted in the production of usherinDexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.
Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5 splice sites (5ss). This interaction did not require branch point recognition and was sensitive to 5ss mutations, suggesting that 48K interacts with the 5ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5ss, as well as functionally important interactions within the U11/U12 di-snRNP.Most metazoans and some unicellular eukaryotes contain two distinct spliceosomes (reference 28 and references therein). The majority of introns are excised by the major, U2-dependent spliceosome, while a subset (Ͻ0.5%) with highly conserved 5Ј splice sites (5Јss) and branch point sequences (BPS) are removed by the minor, U12-dependent spliceosome (for a review, see reference 24). Both spliceosomes consist of five small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins. The two spliceosomes share the U5 snRNP, while the remaining four snRNPs in each spliceosome are distinct but functionally analogous, with U11, U12, U4atac, and U6atac of the minor spliceosome being the counterparts of U1, U2, U4, and U6 in the major spliceosome, respectively (12,14,16,34,35,42).Intron recognition is achieved via multiple, dynamic RNAand protein-mediated interactions. RNA-RNA interactions in the two spliceosomes are highly analogous. In the major spliceosome, the first assembly step (generating the E complex) involves the recognition of the 5Јss by U1, while the nonsnRNP protein factors SF1, U2AF65, and U2AF35 bind to the BPS, the polypyrimidine tract, and the 3Јss, respectively (for a review, see reference 26). During this stage, U1 base pairs with the pre-mRNA's 5Јss, while U1-associated proteins facilitate 5Јss recognition or stabilize the U1-5Јss complex (for a review, see reference 37). During prespliceosome (A complex) formation, U2 associates stably with the BPS (17), while non-snRNP proteins bridge U1 and U2 snRNPs (40, 41). The U4/U6/U5 tri-snRNP then joins the spliceosome (generating the B complex), which triggers the displacement of U1 from the 5Јss by U6 and additional rearrangements that lead to catalytic core formation (for a review, see references 22 and 33).The overall assembly pathway of the minor...
Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12-and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.localization ͉ pre-mRNA splicing ͉ snRNA ͉ U12-dependent spliceosome P re-mRNA splicing is an essential step in the co/posttranscriptional processing of eukaryotic transcripts before their export from the nucleus. The low-abundance U12-dependent ''minor'' spliceosome exists in parallel with the U2-dependent ''major'' spliceosome in most multicellular eukaryotes (1). It catalyzes the removal of a rare class of introns (U12-type) that represent Ͻ1% of introns in mammals (2, 3). The U12-dependent spliceosome contains four unique snRNAs: U11, U12, U4atac, and U6atac, which are paralogs of U1, U2, U4, and U6 snRNAs of the U2-dependent spliceosome, respectively (4, 5). U5 snRNA, in contrast, is shared between the two spliceosomes. In addition to the snRNAs, both spliceosomes contain numerous snRNP and non-snRNP proteins (6, 7).The five snRNP components of the major spliceosome are predominantly localized in the nucleus, but the biogenesis of four of them involves a cytoplasmic step (8). That is, after transcription by RNA Pol II, the snRNAs U1, U2, U4, and U5, which all contain an Sm protein-binding site, are first exported into the cytoplasm. The Sm proteins subsequently bind, and the snRNA's cap is hypermethylated to 2,2,7-tri-methyl-guanosine (m 3 G) (9, 10). These processing steps are a prerequisite for their reimport into the nucleus (11). In contrast to these so-called Sm-class snRNAs, U6 snRNA is transcribed by RNA pol III, acquires Sm-like proteins (Lsm2-8) (12) and a ␥-monomethyl phosphate cap, and does not leave the nucleus. The minor snRNAs U11, U12, and U4atac are also Sm-class snRNAs, containing an Sm-binding site that is also bound by Sm proteins and an m 3 G cap (5, 13), whereas U6atac is transcribed by RNA pol III, possesses a ␥-monomethyl phosphate cap (5), and is assembled with Lsm proteins (14). After nuclear import, spliceosomal snRNPs first pass through Cajal bodies, where they undergo further modifications and assembly (15, 16) and then subsequentl...
The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.
Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.
Alternative splicing (AS) is a major contributor to proteome diversity, but it also regulates gene expression by introducing premature termination codons (PTCs) that destabilize transcripts, typically via the nonsense-mediated decay (NMD) pathway. Such AS events often take place within long, conserved sequence elements, particularly in genes encoding various RNA binding proteins. AS-NMD is often activated by the protein encoded by the same gene, leading to a self-regulating feedback loop that maintains constant protein levels. However, cross-regulation between different RNA binding proteins is also common, giving rise to finely tuned regulatory networks. Recently, we described a feedback mechanism regulating two protein components of the U12-dependent spliceosome (U11-48K and U11/U12-65K) through a highly conserved sequence element. These elements contain a U11 snRNP-binding splicing enhancer (USSE), which, through the U11 snRNP, activates an upstream U2-type 3 ′ ss, resulting in the degradation of the U11-48K mRNA by AS-NMD. Through phylogenetic analysis, we now identify a G-rich sequence element that is conserved in fishes as well as mammals. We show that this element binds hnRNPF/H proteins in vitro. Knockdown of hnRNPH1/H2 or mutations in the G-run both lead to enhanced activation of the 3 ′ ss in vivo, suggesting that hnRNPH1/H2 proteins counteract the 3 ′ ss activation. Furthermore, we provide evidence that U1 binding immediately downstream from the G-run similarly counteracts the U11-mediated activation of the alternative 3 ′ ss. Thus, our results elucidate the mechanism in which snRNPs from both spliceosomes together with hnRNPH1/H2 proteins regulate the recognition and activation of the highly conserved alternative splice sites within the U11-48K pre-mRNA.
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