The natural history of group I introns

There are four major classes of introns: self-splicing group I and group II introns, tRNA and/or archaeal introns and spliceosomal introns in nuclear pre-mRNA. Group I introns are widely distributed in protists, bacteria and bacteriophages. Group II introns are found in fungal and land plant mitochondria, algal plastids, bacteria and Archaea. Group II and spliceosomal introns share a common splicing pathway and might be related to each other. The tRNA and/or archaeal introns are found in the nuclear tRNA of eukaryotes and in archaeal tRNA, rRNA and mRNA. The mechanisms underlying the self-splicing and mobility of a few model group I introns are well understood. By contrast, the role of these highly distinct processes in the evolution of the 1500 group I introns found thus far in nature (e.g. in algae and fungi) has only recently been clarified. The explosion of new sequence data has facilitated the use of comparative methods to understand group I intron evolution in a broader context and to generate hypotheses about intron insertion, splicing and spread that can be tested experimentally.     

Group II and group III introns of twintrons: potential relationships with nuclear pre-mRNA introns

Two new and important features of introns have emerged from analysis of the Euglena gracilis chloroplast genome. One is a new class of introns, designated group III, that may be the closest contemporaries to nuclear pre-mRNA introns. The second is introns that are interrupted by other introns termed twintrons.     

RNA在RNA剪接中的功能：从催化到调控

对非编码RNA功能的认识是后基因组时代的一个研究焦点,本文主要介绍非编码RNA在RNA剪接中的催化和调控功能。在RNA加工过程中,三大类内含子的剪接都是由RNA成员主导。其中Ⅰ型和Ⅱ型内含子能催化自身的切除和外显子连接反应;而核mRNA内含子的剪接则由剪接体里的小核RNA主导。Ⅰ型和Ⅱ型内含子存在于细菌、低等真核细胞和植物的细胞器内;而真核细胞的核编码蛋白质基因内全部是核mRNA内含子,并且其数目随生物体的复杂性而显著升高。一个多内含子前体mRNA通过选择性剪接产生多种,甚至上万种不同的mRNA和蛋白质,对蛋白质组的复杂度和时空表达调控至关重要。选择性剪接调控由剪接调控蛋白特异识别和结合前体mRNA里所富含的顺式RNA调控元件完成的;系统认识这两者之间的对应关系是揭示基因组表达调控网络的一把钥匙。     

USING RDNA GENES TO UNDERSTAND THE ORIGIN AND EVOLUTION OF SPLICEOSOMAL AND GROUP I INTRONS

Ribosomal DNA genes in lichen algae and lichen fungi are astonishingly rich in spliceosomal and group I introns. We use phylogenetic, secondary structure, and biochemical analyses to understand the evolution of these introns. Despite the widespread distribution of spliceosomal introns in nuclear pre-mRNA genes, their general mechanism of origin remains an open question because few proven cases of recent and pervasive intron origin have been documented. The lichen introns are valuable in this respect because they are undoubtedly of a "recent" origin and limited to the Euascomycetes. Our analyses suggest that rDNA spliceosomal introns have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into r RNAs. We propose that the spliceosome itself (and not an external agent; e.g. transposable elements, group II introns) has given rise to the introns. The rDNA introns are found most often between the flanking sequence G (78%) - intron-G (72%), and their clustered positions on secondary structures suggest that particular r RNA regions are preferred sites (i.e., proto-splice sites) for insertion. Mapping of intron positions on the newly available tertiary structures show that they are found most often in exposed regions of the ribosomes. This again is consistent with an intron origin through reverse-splicing. Remarkably, the distribution and phylogenetic relationships of most group I introns in nuclear rDNA genes are also consistent with a reverse-splicing origin. These data underline the value of lichens as a model system for understanding intron origin and stress the importance of RNA-level processes in the spread of these sequences in nuclear coding regions.     

The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction

Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism. Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse on the affinity-purified spliceosome. The spliceosome also catalyzes a hydrolytic spliced-exon reopening reaction as observed in group II introns, indicating a strong link in their evolutionary relationship. We show here that, by arresting splicing after the first catalytic step, the purified spliceosome can catalyze debranching of lariat-intron-exon 2. The debranching reaction, although not observed in group II introns, has similar monovalent cation preferences as those for splicing catalysis of group II introns. The debranching reaction is in competition with the reverse Step 1 reaction influenced by the ionic environment and the structure of components binding near the catalytic center, suggesting that the catalytic center of the spliceosome can switch between different conformations to direct different chemical reactions.     

A glimpse of the catalytic core of a group II intron

A paper in a recent issue of Science describes the first high-resolution structure of part of the catalytic core of a group II intron that will allow more detailed comparisons between the excision of introns by self-splicing group II introns and by nuclear pre-mRNA introns.     

In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.

It was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions.     

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.     

Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation

Group II intron bl1 from yeast mitochondria can undergo self-splicing in vitro. Exons become correctly ligated, and the excised intron has a lariat structure similar to that of introns from nuclear mRNA. The branch point of the bl1 lariat is located eight or nine nucleotides upstream of the 3' end of the intron and is part of a hairpin structure that is well conserved among group II introns. Several mutations next to the branch point and in other parts of the core structure of group II introns are shown to affect lariat formation. One of them, carried by strain M4873, abolishes splicing in vivo and in vitro, apparently by changing the architecture of the hairpin structure containing the branch point. Similarities between group II introns and nuclear pre-mRNA introns are discussed in terms of evolutionary relatedness.     

Disparate Evolution of Paralogous Introns in the Xdh Gene of Drosophila

Drosophila nuclear introns are commonly assumed to change according to a single rate of substitution, yet little is known about the evolution of these non-coding sequences. The hypothesis of a uniform substitution rate for introns seems to be at odds with recent findings that the nucleotide composition of introns varies at a scale unknown before, and that their base content variation is correlated with that of the adjacent exons. However, no direct attempt at comparing substitution rates in introns seems to have been addressed so far. We have studied the rate of nucleotide substitution over a region of the Xdh gene containing two adjacent short, constitutively spliced introns, in several species of Drosophila and related genera. The two introns differ significantly in base composition and substitution rate, with one intron evolving at least twice as fast as the other. In addition, the substitution pattern of the introns is positively associated with that of the surrounding coding regions, evidencing that the molecular evolution of these introns is impacted by the region in which they are embedded. The observed differences cannot be attributed to selection acting differently at the level of the secondary structure of the pre-mRNA. Rather, they are better accounted for by locally heterogeneous patterns of mutation. Received: 26 July 1999 / Accepted: 21 August 1999     

Chloroplast group III twintron excision utilizing multiple 5''- and 3''-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.     

The splicing of transposable elements and its role in intron evolution

Recent studies have demonstrated that transposable elements in maize and Drosophila are spliced from pre-mRNA. These transposable element introns represent the first examples of recent addition of introns into nuclear genes. The eight reported examples of transposable element splicing include members of the maize Ac/Ds and Spm/dSpm and the Drosophila P and 412 element families. The details of the splicing of these transposable elements and their relevance to models of intron origin are discussed.     

Nuclear pre-mRNA processing in plants: distinct modes of 3''-splice-site selection in plants and animals.

The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.