Mechanisms for intron loss and gain

           This fifth article, Evolution of the mechanisms of intron loss and gain in the social amoebae Dicytostelium, by Ma et al. looks at introns in a fashion completely different from the previous articles in this blog. The authors wanted to explore the mechanisms of intron loss and gain through Dictyostelium, particularly Dictyostelium discoideum and Dictyostelium purpureum. The authors chose Dictyostelium because it breaks away from the typical mold of eukaryotes studied for introns and it has characteristics that make it useful for studying intron evolution. The first characteristic is the amount of simple sequence repeats (SSRs) and the second being 16 new genes Dictyostelium has acquired from bacteria by horizontal gene transfer.

            The authors wanted to test three possible models from the mechanisms of intron gain or loss. The first is the reverse transcription model, in which introns are deleted from the genome by recombination of cDNA and genomic DNA. The second model is simple genomic deletion, which introns are lost regardless of location on the gene. The third model is one where introns are lost during non-homologous end joining repair of DNA.

            After experimentation, the authors found Dictyostelium discoideum had 441 intron losses and 40 intron gains and Dictyostelium purpureum had 202 intron losses and 58 intron gains. These observations maintain the simple genomic deletion theory but do not support the homologous end joining model for intron loss. Next they wanted to figure out why there was more itron loss in one but not the other. Two explanations based on reverse transcription were tested. The first is that D. discoideum had shorter introns and more suitable for recombination. The second is that D. discoideum had higher reverse transcriptase activity than D. purpureum. The second explanation held up as D. discoideum has many more reverse transcriptase genes than D. purpureum.

            The authors also cited how natural selection can show the variations in intron loss and gain. The two species of Dictyostelium separated hundreds of million years ago meaning that that natural selection selected differently for the unique characteristics of each Dictyostelium.

            The explanation for the intron loss mechanism is explained and studied thoroughly the mechanism for intron gain is cloudier. Finding the source sequences for intron has been incredibly difficult as another study was only able to locate one of seven intron gain source sequences. A possibility for this difficulty is that viruses may have contributed exogenous sequences onto the Dictyostelium, this could explain intron gains without having that sequence in the original DNA strand.

            The authors finished their study by stating that the mechanisms for intron loss are very similar to the mechanisms found in animals and fungi. The intron loss found in Dictyostelium was due to the genomic conversion between genomic DNA and cDNA reverse transcribed from mature mRNA.

            This journal successfully provided mechanisms for intron loss but not for intron gains. Despite the intron gains, this study was a success. It was interesting to see how the authors tied in the mechanisms with natural selection to better explain their findings along the results of their experimentation.

Source

Ma, M., Che, X., Porceddu, A., & Niu, D. (2015). Evolution of the mechanisms of intron loss and gain in the social amoebae Dictyostelium. BMC Evolutionary Biology. 15: 286.

Introns and GC-content

After the thorough examination of introns and their splicing effects, this journal, Introns structure patterns of variation in nucleotide composition in Arabidopsis thaliana and rice protein-coding genes, by Ressayre et al. explores how introns can influence the nucleotide composition (GC content). As the title suggests the authors looked at two plants species that that offered the best annotated gene structures among plants with A. thaliana being GC-poor and rice being GC-rich, respectively.

The authors wanted to investigate the potential role of introns in GC-content variation in plant genes. They focused on the link between intron presence and GC-content. They observed tight links between intron presence and variation in GC-content into a negative correlation between intron number and GC-content. The intron/exon structure was shown to effect nucleotide, codon, and amino acid compositions.

The results of their experimentation and numerous calculations revealed that introns impede the increase in GC-content. They found this by comparing intron infused in 5’ UTR and 5’ UTR without introns. They found that the genes with the introns were smaller compared to the genes without the introns.

In the experiment the authors looked at internal and external coding regions and how they interacted introns. Introns appear to delimit gene space in three different regions, one internal and the other external. They observed lower variation in GC-content in internal sequences, this resulted in a more complex pattern along the genes with negative correlations. Gene intron number is expected to be a strong determinant of CDS GC-content. The introns have more effect in GC-rich regions but have less of an effect in GC-poor regions.

The authors concluded that gene intron number and precise gene-intron architecture are required to properly describe patterns of variation in GC content. The GC-content is correlated with many genomic features such as recombination rates, splicing mechanisms, and nucleosome positioning. They propose that the effects of introns on the GC-content have a long-range effect on those genomic features. They suggest that an intron barrier exists that explains the peculiarities of GC-content. The intron acts as barriers in recombination or extension of conversion for external GC-content. This intron barrier can increase the GC-content of external genes while acting as a constraitn on internal GC-content. In regards to internal GC-content the introns can suppress the increase in the spreading. While the presence of introns has effects on GC-content without the introns the GC-content would be more susceptible to evolutionary change that could negatively impact the organism.

The previous journal articles have discussed how introns can be beneficial via splicing and regulation. This one looked at how the physical presence of introns affected the architecture of the GC-content in these plants, the authors mentioned other plants and how their architecture varies to produce variants of the genes studied. From these journals I see how the introns have a legitimate influence in the structure and expression of genes. Much smaller compared to exons but enough to have a positive or negative impact.

Sources:

Ressayre, A., Glemin, S., Montalent, P., Serre-Giardi, L., Dillmann, C., & Joets, J. (2015). Introns structure patterns of variation in nucleotide composition in Arabidopsis thaliana and rice protein-coding gene. Genome Biology and Evolution. PMCID: PMC4684703

Splicing in Human Genes

            Intron splicing discussed in the previous article was on a whole new level for me. Chasing some links under “similar articles” I found this journal, which focuses on the splicing of introns in regards to human genes. Since this article deals with human genes it hits closer to home so let’s get started.

           

            Splicing of many human geens involves sites embedded within introns by Kelly et al., want to challenge the old ways of looking at splicing and open the door for a broader sense of what splicing is. The conventional method of splicing and introns, regarded as “junk”, that they want to question is the that the 3’ end of one exon directly connects to the 5’ prime end of another exon with the introns completely removed from the equation. They also explore how genes yield products generated by intermediate intron splicing and how inhibiting the intermediate splicing prevents efficient exon-exon splicing.

            The diagram below shows the differences between the conventional model versus the recursive model that the authors experiment with.

             As mentioned before, the conventional model has 3’ end of one exon (donor) meet with the 5’ end of another exon (acceptor) joined together. The recursive model has the 3’ end join to sites within the intron, RS1 and RS2, before combining with the 5’ end. What the diagram is trying to show is how introns removal assist in the completion spliced sequence. Having the 3’ end of the exon connect with RS1 and RS2 it increases the splicing efficiency by bringing the 3’ end to the 5’ end in sequential steps rather than one big jump. A big jump could cause errors or difficulties due to the intron’s length, which is very long in human genes.

            Continued experimentation revealed that mutations of the RS sites negatively affect the amount of mature exon-exon products. The diagrams below demonstrate the effects of mutations.

           

            These diagrams show a series of events of how the RS sites can be mutated and no longer functioning. The comparison was done between the mutants and wild types. The exons themselves are maintained but the completed splice and mature mRNA decrease due to the lack of RS sites present.

            At the end of their experimentation the authors concluded that the introns and their RS sites directly correlate with exn-exon splicing and the production of mature mRNA. They also state that the RS sites can act as regulatory sites to limit the overproduction of certain proteins. The exact mechanisms of the RS sites and their influence on splicing remains unclear and further studies are needed.

            The experimentation done by the authors on human genes made this journal more interesting by demonstrating that nothing in the DNA or the body is junk. The cost-benefit analysis on introns thus far reveals that introns are an important factor in regulation and gene splicing. Despite initial concerns of the amount of DNA sequence that they take up. Learning more and more about introns is flipping some of my fundamental ideas of DNA and gene expression upside down. The introns, promoters, poly-A tails, and so on show me how much of the DNA is used to actually get those genes through the process of transcription and translation.   

Sources:

Steven, K., Georgomanolis, T., Zirkel, A., Diermeier, S., O’reilly, D., Murphy, S., Langst, G., Cook, P.R., & Papantonis, A. (2015). Splicing of many human genes involves sites embedded with introns. Nucleic Acids Research. 43: 4721-4732.

Introns and Splicing

Continuing a theme from the last post, this next article, Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes, by Petibon, Parenteau, Catala, and Elela examined the mechanism of how introns regulate the expression of dRPGs (duplicate ribosomal protein genes) and how it relates to the synthesis of ribosomal proteins (RPs). The article shows how introns can determine the ratio of ribosomal protein isoforms through regulation of splicing. The authors used yeast, Saccharomyces cerevisiae, as their medium for this study.

           In the introduction, the authors demonstrated explain how ribosome synthesis is a major undertaking that requires precise coordination without wasting energy or compromising quality. They continue with how the duplication of genes and deletions of certain duplicates can generate different transcription profiles and affect growth. Introns become a part of this dRPG process in defining the expression pattern. The initial idea was that introns lead to only inhibition of gene expression. However introns induced the expression of about half of the genes and inhibited the expression of the other half meaning that introns may act as gene-specific regulators.

 To test their hypothesis, the yeast was grown and manipulated using standard procedures. They tagged RPS9 genes as RPS9 genes can be introduced with intron deleting strains. They used to variants called RPS9A and RPS9B which acted as paralogs of each other. Going through the methods of the experiment the authors were able to observe the changes in RNA, proteins, splicing, and growth of the yeast form the various tests conducted.

            They found that introns play an important role in controlling or coordinating the expression of RPGs but the mechanism used to fluctuate the RPG expression is another mystery. The authors found that the intron is preferred tool for repression of the RPS9A gene and that the deletion of the intron allows for a greater expression of the RPS9A gene.  The splicing of the RPS9A and RPS9B was also examined and the results showed that splicing efficiency was greater when the intron was present.

            To test how splicing efficiency affected the production of mRNA and proteins, the authors looked at the secondary structures. By performing intron substitution on the RPS9 genes, these substitutions increased mRNA levels. They concluded that the expression levels are influenced by the intron structure and splicing. This study showed how the preservation of introns in yeast is crucial for gene regulation via gene splicing.  

            Going through this study made me realize that there is a lot more to gene splicing and regulation than I had originally thought. I was under the impression that gene splicing was relevant to expressing slight variation in proteins through exons but it turns out that introns are as equally involved in splicing. I have to give credit ot the teachers at OCC for making this topic easier to understand in the classroom otherwise it would be completely overwhelming. 

Sources:

Petibon, C., Parenteau, J., Catala, M., & Elela, S.A. (2016). Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes. Oxford Journals. 44: 3878-3891.

What's the purpose of Introns?

In the various biology courses I have taken at OCC there have been many things that I have found fascinating, confusing, and what felt like straight out of science. One continuous theme throughout those classes was that everything has a reason and that nothing goes to waste.

When the topic of DNA transcription and translation came about one aspect always stood out, introns. The way I studied for a test was that exons were expressed and introns were thrown away. Outside of the test, I could not actually believe that introns were meaningless. When this semester project came around I thought it would be a good idea to tackle this question and see what I can learn.

After searching through pubmed looking for articles, I stumbled upon this article by Jo and Choi called Introns: The Functional Benefits of Introns in Genomes. This was the perfect article to break the ice and dive into the purpose of introns.

Jo and Choi conducted their research to understand the functional roles or benefits of introns. They start by breaking introns into two possible roles: direct or indirect.

            To begin their explanation of introns with direcr roles, they explore how introns are a part of the regulation needed for alternative splicing and gene expression. Alternative splicing produces variant proteins from a single gene. They suggest that introns carry cis-acting elements that allows for the conservative exons to be properly expressed.

            As for the gene regulation, they looked at research Buchman and Berg whose study revealed that proteins produced by simian virus with introns were 400 times greater than simian virus without introns. It showed how introns guarantee a higher level of expression. This idea was termed intron-mediated enhancers (IME). In fact, gene expression increased with IMEs in studies conducted with plants and mammals.

            The authors continue the direct roles of introns in the processes of transcription and translation. IMEs can regulate transcription by modulating the functions of promoters on genes. Introns also contribute to the mRNA transport outside of the cell to have the mRNA translated in the cytoplasm.

            The authors shift gears and explore the indirect roles of introns. They examine how introns can influence creating new genes and natural selection. In the creation of new gens that cited an experiment conducted by Carvunis et al. that looked at proto-genes, non-functional translated code, and how they could become functional genes. The authors infer that introns could be a part of these porot-genes and could lead to new genes due to mutations and alternative splicing.

            In natural selection, introns increase the efficiency of natural selection via recombination and the relaxation of Hill-Robertson (HR) interference. The introns can reduce the HR interference by increasing the recombination possibilities for favorable exons that can lead to new and better genes. Introns can also act as a mutation buffer allowing for the allele to be expressed properly with causing harm to the organism.

            The authors conclude their findings by stating that introns do have functional roles in the cell and organism and that further research is needed to uncover all of the possible roles of introns. One other point that is discussed is the energy cost of copying introns when they may take up to 40% of a gene’s code. But keeping the introns may be in the cell’s best interest due to the direct and indirect roles introns have.

After reading through that first article, I am pleased to learn that introns are in fact not useless and can have a substantial impact on the life of a cell or organism. Now I know that I need to dig deeper to learn more.  Let’s see what I can find next.

Sources:

Jo, B. & Choi, S.S. (2015). Introns: The Functional Benefits of Introns in Genomes. Genomics and Informatics.

Buchman AR, Berg P. (1988). Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 8:4395–4405

Carvunis AR, Rolland T, Wapinski I, Calderwood MA, Yildirim MA, & Simonis N. (2012). Proto-genes and de novo gene birth. Nature. 487:370–374.