Why do introns need to be removed

The sequence and length of introns vary rapidly over evolutionary time. Scientists have found clear examples of 'functional nuclear introns' that can accommodate sequences important for the expression of the gene on which the intron resides.

This function is not a general feature of introns, however, because several genes that lack introns express themselves normally histones and olfactory receptor genes, for instance. There are also cases in which introns contain genes for small nuclear RNA, which is important for the translation of messenger RNA, an intermediary between DNA and proteins.

Nuclear introns can also be important in a process called alternative splicing, which can produce multiple types of messenger RNA from a single gene.

Although these examples demonstrate a constructive role for introns, they cannot explain why introns are so ubiquitous in our genes. He suggested that introns could speed up evolution by promoting genetic recombinations between exons.

This process which he called 'exon shuffling' would be directly associated with formation of new genes. Introns, from this perspective, have a profound purpose. They serve as hot spots for recombination in the formation of new combinations of exons. In other words, they are in our genes because they have been used during evolution as a faster pathway to assemble new genes.

Over the past 10 years, the exon shuffling idea has been supported by data from various experimental approaches. They are expected to yield a huge amount of information about intron sequences.

The new data should solve most of our basic questions about the functions of introns. Newsletter Get smart. Sign up for our email newsletter. A more straightforward NGS analysis of the splicing intermediates confirms that either of these two introns can be removed as the first.

Nonetheless, many more reads correspond to transcripts, from which only the first intron i1 was removed Fig 3C ; amplicon i5-tubA, groups II and IV— vs. Among the NGS reads representing the transcripts for the i6-tubA amplicon, all possible combinations of occurrence of conventional introns can be noticed: reads with single introns such as i1, i2 or i5 removed groups VI, X and III; Fig 3B , with pairs of introns removed i1 and i2, group V; i1 and i5, group II; i2 and i5, group VII; Fig 3B and reads with all three introns removed i1 and i2 and i5, group I; Fig 3B.

This shows the lack of a strict order for conventional intron removal from the tubA transcript and implies a moderate preference for their excision.

It seems that intron i5 nt long is preferentially removed as first reads with i5 primarily removed; Fig 3B , group III , followed by short intron i1, which is 52 nt long reads; Fig 3B , group VI and finally, the longest of them, intron i2, having nt only two reads; Fig 3B , group X.

The band pattern seen on the agarose gel comparing the relative order of nonconventional intron removal is quite unexpected Fig 1E and 1F ; pairs C, G and H. In all three cases, only the product corresponding to the transcript maintaining both introns was observed, and the product with solely a single intron removed was not detected. Such a picture indicates that the abundance of nonconventional splicing intermediates is very low. Results of sequencing performed for the amplicons confirm this observation.

In the case of gapC and i6-tubA, a high level of reads with unprocessed nonconventional introns is present gapC: 11, reads, i6-tubA: 2,; Fig 3A and 3B. Reads with all analyzed nonconventional introns removed are also detectable gapC: 17 reads, i6-tubA: 9 reads; Fig 3A and 3B , which suggests that using the primer corresponding to the last intron in the reverse transcription reaction to exclude mature mRNA from the analysis was not the main factor limiting the pool of splicing intermediates.

However, in this regard results obtained for amplicons should be treated with consideration. Meanwhile, barely eight reads with the single nonconventional intron removed were identified across the studied amplicons i4 in i6-tubA, group IX; no reads were found without i2 or i3 in gapC and i3 in i6-tubA; Fig 3A and 3B.

This number is extremely low when compared to the level of existing intermediate forms of conventional splicing, which were, in fact, very abundant more than thousands of them among all amplicons and diversified. The low level of nonconventional splicing intermediates observed in both performed experiments may suggest that nonconventional splicing not only begins later than the conventional one, but also takes place more rapidly.

However, the low level of nonconventional splicing intermediates may also be the effect of a coordinated start of the removal process of all nonconventional introns of quite similar length from the single transcript. Multiple types of transcripts at various stages of pre-mRNA maturation were detected as a result of the analysis of the intermediate splicing products. Numerous reads corresponding to immature transcripts containing all introns are observed among the gapC and i6-tubA amplicons.

The same applies to reads corresponding to various splicing intermediates as well as reads representing transcripts without analyzed introns. However, a strikingly high percentage of reads equivalent to transcripts with all conventional introns removed and all nonconventional present aroused attention.

In the case of the gapC amplicon there are as many as 11, of such reads Though it is worth mentioning that the methodology adopted in the experiment was based on the amplification procedure, implying that the number of obtained reads does not necessarily directly reflect the number of intermediates present in the cell.

Nevertheless such a picture may indicate that these types of transcripts accumulate before the dynamic nonconventional splicing begins. This, in turn, may suggest existence of some factor delaying the initiation of nonconventional intron removal. It cannot be ruled out that the different localization of both types of splicing may play a role here.

Conventional splicing is tightly coupled with transcription—it starts before a pre-mRNA molecule synthesis completion. Perhaps nonconventional splicing cannot launch at such a stage maybe due to the lack of splicing factors? Moreover, it should not be ruled out that nonconventional splicing may take place much later, even in the cytoplasm. However, in order to confirm this hypothesis, more insightful research would have to be done in the future.

In this article, we summarize a series of experiments determining the order of processing of conventional and nonconventional introns, carried out for E. This constitutes another portion of knowledge about atypical nonconventional introns, a research area still extremely challenging due to the lack of well-established methods.

Further studies will be needed to answer some of the remaining questions. What is the mechanism of atypical nonconventional intron removal?

Is the theory of their transposon origin correct? Likewise, how may their propagation in nuclear genomes be related to the activity of DNA repair systems as it was hypothesized previously [ 22 ]. Cells were cultured in the Cramer-Myers medium [ 31 ], supplemented with 0. This procedure was performed according to the rigorous digestion conditions described in the manufacturer's instructions.

The integrity of the RNA sample was also checked via electrophoresis under denaturing conditions 0. The amount of 0. In each case, negative controls which consisted of reaction mixtures without the addition of a reverse transcriptase, were also performed.

PCR products were sized on 1. Sequencing reads were initially quality controlled and trimmed by the sequencing institution. In addition, a FastQC 0. Afterwards, sequencing reads were filtered manually, based on the sequences of primers used for amplification.

Each of the gapC and tubA contigs was mapped using Geneious mapper algorithm against the reference sequences of gapC GenBank accession number: L Only successfully mapped, complete reads—flanked by PCR primers at both ends which indicates complete PCR product amplification were taken into consideration in the further analysis. Incomplete reads without the sequence s of primers , as well as reads extending beyond the primer binding site s were discarded.

Remaining fair reads were subsequently compared and categorized based on the presence or absence of individual intron s in the obtained alignment. Mapped sequences were then sorted based on the alignment length. Afterwards, all possible read types for both studied genes were annotated and counted. The most probable order of intron removal has been inferred from the summarized statistics of the reads. Schematic depiction of products obtained with each primer set for both studied genes B, D.

Arrows represent primers, lines represent PCR products. Gaps within spliced products horizontal dotted lines refer to the absent intronic sequences.

Product lengths are given alongside depictions. We would like to thank dr Anna Karkowska for providing us with the access to the Geneious Basic Abstract Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes.

Author summary The existence of conventional spliceosomal introns in genes of eukaryotic organisms is a well-known theorem. Introduction Nuclear genes of eukaryotes contain introns which are removed from pre-mRNA in a splicing process catalyzed by the spliceosome—a ribonucleoprotein complex assembled from five small nuclear RNAs snRNA and a range of proteins.

Download: PPT. Order of outron and first intron removal in the rbcS transcript Results obtained for tubA and gapC transcripts contradicted the data in the literature which indicates that nonconventional splicing occurs before the conventional one [ 26 ]. Fig 2. Order of removal of the outron and the first intron in the rbcS transcript.

Fig 3. Summarized statistics of sequencing reads As a result of sequencing, raw PacBio reads of various lengths corresponding to three abovementioned amplicons were received. Categories of sequencing reads The gapC amplicon produced three possible categories of sequencing reads, with: I the first conventional intron absent, II all introns present, and III all analyzed introns spliced out.

Order of conventional introns removal in tubA transcripts The only example which can allow for tracking the possible order of excision of conventional introns, is provided via the tubA gene, where the two conventional introns are located next to each other. The pace of nonconventional splicing The band pattern seen on the agarose gel comparing the relative order of nonconventional intron removal is quite unexpected Fig 1E and 1F ; pairs C, G and H.

Time localization? Outline In this article, we summarize a series of experiments determining the order of processing of conventional and nonconventional introns, carried out for E. Supporting information. S1 Table. List of the primers used in this study. Primers used to generate amplicons are marked with an asterisk. S1 Fig. Acknowledgments We would like to thank dr Anna Karkowska for providing us with the access to the Geneious Basic References 1. Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription.

Pre-mRNA processing reaches back to transcription and ahead to translation. Co-transcriptional splicing of constitutive and alternative exons. Singh J, Padgett RA.

Rates of in situ transcription and splicing in large human genes. Nat Struct Mol Biol. Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Mol Cell. Neugebauer KM. On the importance of being co-transcriptional. J Cell Sci. Aebi M, Weissman C. Precision and orderliness in splicing. Trends Genet. View Article Google Scholar 9. Nucleic Acids Res.

Outcome of donor splice site mutations accounting for congenital afibrinogenemia reflects order of intron removal in the fibrinogen alpha gene FGA. Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA. The Past and Future of Introns. References and Recommended Reading Berget, S. Cell 12 , 1—8 Darnell, J.

Cell 20 , — Knapp, G. Cell 14 , — Konarska, M. Genetics: A Conceptual Approach , 2nd ed. New York, Freeman, Roy, S. Article History Close. Share Cancel. Revoke Cancel. Keywords Keywords for this Article. Save Cancel. Flag Inappropriate The Content is: Objectionable. Flag Content Cancel.

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