Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs

Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3′ and 5′ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5′ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

Alternative splicing and bioinformatic analysis of human U12-type introns

U12-type introns exist, albeit rarely, in a variety of multicellular organisms. Splicing of U12 intron-containing precursor mRNAs takes place in the U12-type spliceosome that is distinct from the major U2-type spliceosome. Due to incompatibility of these two spliceosomes, alternative splicing involving a U12-type intron may give rise to a relatively complicated impact on gene expression. We studied alternative U12-type intron splicing in an attempt to gain more mechanistic insights. First, we characterized mutually exclusive exon selection of the human JNK2 gene, which involves an unusual intron possessing the U12-type 5′ splice site and the U2-type 3′ splice site. We demonstrated that the long and evolutionary conserved polypyrimidine tract of this hybrid intron provides important signals for inclusion of its downstream alternative exon. In addition, we examined the effects of single nucleotide polymorphisms in the human WDFY1 U12-type intron on pre-mRNA splicing. These results provide mechanistic implications on splice-site selection of U12-type intron splicing. We finally discuss the potential effects of splicing of a U12-type intron with genetic defects or within a set of genes encoding RNA processing factors on global gene expression.     

Mutational analysis of the U12-dependent branch site consensus sequence

Highly conserved sequences at the 5′ splice site and branch site of U12-dependent introns are important determinants for splicing by U12-dependent spliceosomes. This study investigates the in vivo splicing phenotypes of mutations in the branch site consensus sequence of the U12-dependent intron F from a human NOL1 (P120) minigene. Intron F contains a fully consensus branch site sequence (UUCCUUAAC). Mutations at each position were analyzed for their effects on U12-dependent splicing in vivo. Mutations at most positions resulted in a significant reduction of correct U12-dependent splicing. Defects observed included increased unspliced RNA levels, the activation of cryptic U2-dependent 5′ and 3′ splice sites, and the activation of cryptic U12-dependent branch/3′ splice sites. A strong correlation was observed between the predicted thermodynamic stability of the branch site: U12 snRNA interaction and correct U12-dependent splicing. The lack of a polypyrimidine tract between the branch site and 3′ splice site of U12-dependent introns and the observed reliance on base-pairing interactions for correct U12-dependent splicing emphasize the importance of RNA/RNA interactions during U12-dependent intron recognition and proper splice site selection.     

Alternative Processing of Primary microRNA Transcripts by Drosha Generates 5′ End Variation of Mature microRNA

Background

It is generally believed that the miRNA processing machinery ensures the generation of a mature miRNA with a fixed sequence, particularly at its 5′ end. However, we and others have recently noted that the ends of a given mature miRNA are not absolutely fixed, but subject to variation. Neither the significance nor the mechanism behind the generation of such miRNA polymorphism is understood. miR-142 is an abundantly expressed miRNA in hematopoietic cells and exhibits a high frequency of 5′ end polymorphism.

Methodology/Principal Findings

Here we show that a shift in the Drosha processing of pri-miRNA generates multiple forms of miR-142s in vivo with differing 5′ ends that might target different genes. Sequence analysis of several pre-miRNA ends cloned from T cells reveals that unlike many other pri-miRNAs that are processed into a single pre-miRNA, pri-miR-142 is processed into 3 distinct pre-miR-142s. Dicer processing studies suggest that each of the 3 pre-miR-142s is processed into a distinct double-stranded miRNA, giving rise to 4 mature miRNA variants that might regulate different target gene pools.

Conclusions/Significance

Thus, alternative Drosha processing might be a novel mechanism for diversification of the miRNA target gene pool.     

Complex splicing control of the human Thrombopoietin gene by intronic G runs

The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1–4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3′ splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3–5 guanosines (namely, G1–G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7–G10 work in a combinatorial way to control the selection of the proper 3′ splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3′ splice site so providing functional evidence that this factor is involved in the selection of the authentic 3′ splice site of THPO IVS2.     

Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing

Using site-specific incorporation of the photochemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3′ splice sites. One of these is an unidentified ~122 kDa protein that cross-links to 3′ splice sites during formation of the pre-spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3′ splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3′ splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3′ splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3′ splice site of yeast introns is contacted much earlier than previously envisioned by a protein of ~120 kDa, and that a transient association of Prp22p with the 3′ splice site occurs between the first and second catalytic steps.