Investigating Intron-Mediated Enhancement in Caenorhabditis elegans, Summaries of Biology

Download Investigating Intron-Mediated Enhancement in Caenorhabditis elegans and more Summaries Biology in PDF only on Docsity!

Enhancement of transgenes through Intron-Mediated Enhancement in Caenorhabditis elegans BIT May 29, 2009

Abstract

Introns can facilitate the evolution of new proteins through exon shuffling and alternative splicing. Introns have also been observed to regulate genes, through the role of nonsense-mediated mRNA which leads to degradation of the mRNA and Intron-mediated enhancement (IME). As seen in many organisms, IME has a positive effect on gene expression. Introns must be located within the transcribed sequence to elevate the mRNA accumulation in plants and mammals. Although, Introns are known to enhance gene regulation through the mechanism of IME, the results of variation in position of the intron within the transgene is unknown in many organisms. This proposal will explore if variation in intron positioning effects gene enhancement in C. elegans and strive towards further understanding of IME. Reporter gene constructs with intron insertion at 5’ position and 3’ position will be introduce into C. elegans as single-copy transgenes, and the effects of intron position on reporter gene expression will be analyzed. A better understanding of IME and the affects of different intron position on enhancement and regulation will aid in understanding how genes are regulated and allow for better design of transgenes.

Table of Contents

I. Introduction

a. Biological Question Gene expression is regulated at many levels through numerous mechanisms. One of these highly influential mechanisms is intron mediated enhancement (IME), a process by which introns have a positive effect on gene expression. IME is observed in many organisms, but it is the least understood area of gene expression in Caenorhabditis elegans as well as other organisms. This proposal will explore if intron position is important and determine if IME decreases with distance from the start of transcription as discovered in Arabidopisis.

b. Background Introns are often needed for full expression of genes in multiple organisms. Introns can significantly affect gene expression in plants and many other eukaryotes in a variety of ways. Some introns contain enhancer elements or alternative promoters, while many others elevate mRNA accumulation by IME. In other eukaryote species the introns involved in IME must be within transcribed sequences near the start of a gene and in their natural orientation to increase expression. The features of intron-mediated enhancement (IME) are mechanistically the same in many observed species; the introns must be located within the transcribed sequence to affect the mRNA accumulation in plants and mammals. Although, introns are known to enhance gene regulation through the mechanism of IME, the sequences of these introns are unknown it is highly variable what these intron sequences consist of because you can delete parts of introns and still have IME, this suggests that there are multiple and dispersed sequences.. Such a conclusion is reached because not all introns affect gene expression and/or not all genes contain enhancing introns.

The effects of gene expression vary by the position of the intron. Most transgenes that are naturally expressed at high levels contain introns and are considered of having a lot more enhancing signals comparing to poorly expressed genes (Rose, A. B., 2008). Six different locations within a gene were tested individually in Arabidopisis and mRNA accumulation of the transgene was found to decline as the intron positioning is moved further away from the promoter towards the 3’ end. When the intron was placed 1.1 kb or further away from the transcription site, the intron did not enhance the transgene (Rose, A. B., 2002). Similarly, introns placed downstream of the stop codon fail to enhance in mammalian studies. Even though, there are plenty of gray areas in the study of IME one model that is consistent with all the data available, is that the intron sequences found in 5' introns, arbitrate a change in the transcription machinery which results in a much more stable transcription and translation. When IME signals are absent,

RNA polymerase is likely to dissociate and leave immature mRNA to be degraded by exosomes. Enhancement of tensgene is directly linked with the presence of intron at the 5’ end of a given transgene (Rose et al, 2008).

An effect of introns on gene expression was also seen in C. elegans although not studied in great detail. General effects were seen with synthetic introns at 5’ position in C. elegans. First intron (known as promoter-proximal) and third intron in specific genes (myo-3 and unc-54) of C. elegans acted as the enhancers, also large introns placed at the 5’ position led to activation of enhancement. The introns are also associated with mRNA splicing which is another form of regulation possibly by IME (Okkema et al, 1993). the synthetic intron also enhanced but the enhancer that Okkema found in the C. elegans was a specific enhancer element in one intron of unc-54 which is not the same as the general intron effect they found; they found a body muscle enhancer and then a general effect of other enhancers. Okkema thought splicing was causing such an effect on IME because the synthetic intron worked whereas later Rose’s paper demonstrated that splicing per se is not required for IME, although there may be an association of the splicing machinery in the process. IME may therefore serve an important role in mRNA surveillance for real introns. Further studies of the introns in C. elegans can result in fruitful results for researchers who are interested in maximizing gene expression from a recombinant construct-by inserting introns that are known to enhance expression. One may even be able to design synthetic introns that outperform natural

c. Hypothesis

Placement of an intron at the 5’ position of the transgene, but not the 3’ position, will result in IME in C. elegans as observed in Arabidopsis and other species. Through the knowledge gained researchers worldwide can use specific intron positioning to their advantage for enhancement or regulation of transgene.

II. Specific aims

a. Objective 1 To find out if intron-mediated enhancement is position dependent in Caenorhabditis elegans and to understand if the mechanism of IME is the same in Caenorhabditis elegans as seen in plants and mammals.

III. Rational and Significance

Introns can significantly affect gene expression in plants and many other eukaryotes in a variety of ways. Some introns contain enhancer elements or

gene (pELS3) (Okkema et al, 1993). These constructs were generated in pBS by Dr. Alan Rose.

pELS

pELS

pELS

Construct were inserted in the C. elegans through bombardment of the plasmids and then expression was scored through GUS staining. Such a protocol was used to obtain qualitative data because with bombardment we do not get an integrated single copy insertion and the staining (intensity of the blue color) could be affected by the differentiating number of the transgene copies present in the genome therefore, we will use the Mos transposon to assure that we attain a single copy insertion. IV. Experimental methods and procedures

We obtain a single copy line of our plasmid through the injection protocol and stain the progeny to observe the expression of different constructs. I will start with the gene previously shown to exhibit intron mediated enanchement, unc-54. I will generate constructs that allow me to test whether intron position is important (Okkema et al, 1993).

These constructs will then be subcloned with pCF151 vector, which allows for transformation into C. elegans. This vector includes unc-119 +^ marker with Mos transposon sequence and will be injected into unc-119 -^ worms that have a Mos target site. A single copy of the plasmid will then get integrated into the genome of the c. elegans at the Mos target site (transformation and Mos Paper). transgenic integrated lines will be identified by look for unc-119 +^ progeny of the injected C. elegans, and then homozygous lines will be generated.

10 L4 (young adults) will be placed in a 4x6 multiwell dish in .4ml of .6% SDS to permeabilze them. The worms will be rinsed and stained then with X-gluc staining buffer (Okkema et al, 1993). The blue color produced by the GUS conversion of X-gluc will be scored as to intensity of color and time of appearance. Based on our previous analysis of X-gluc stained worms, we will for example, assign3+ for staining that appears blue within 2-4 hours, dark blue by 24

hours, and still dark blue by 48 hours; assign2+ for staining that appears to have no change in color within 2-4 hours, some color by 24 hours, and medium blue by 48 hours; assign1+^ for staining that appears to no color up until 24 hours and faint blue by the 48 th^ hour (observation of similar constructs in lab). The intensity of the color will quantify how much of the gene is expressed, the faster the blue stain appears it will indicate how much more the gene was expressed. We will test two lines for each of the three constructs because with the Mos transposon we are sure that we attained a single copy transgene at the unc-119-^ with Mos target site. If we observe the accepted results then RTPCR will be used to confirm the pervious data quantitatively (Frokjaer-Jensen, 2008).

Nontransformed unc-119-^ will be used as a negative control because this is an unc-119-^ transgene with out GUS so it will not stain at all. pELS2 (intron from Okkema et al, 1993) will be used as the positive control because it expresses unc- 119 -^ with GUS and also because we already know this work from our previous experience with the qualitative assay. We predict that pELS6 (intron positioned at the 3’ end) will be as faint as pELS1 (no intron) and much lighter than pELS2. Such results will indicate that position is important for enhancement in gene expression. Strong expression with the intron at the 3’ position will conclude that the IME is reversed for C. elegans or that the intron positing does not affect the expression of a gene. Knowledge gained from this experiment will enable us to design better transgenes for use in the labs' studies, and understand if the mechanism of IME is the same in Caenorhabditis elegans as seen in plants and mammals.

Our back up plan if the Mos transformation does not work will be to go back to the bombardment method which can give integrated and extrachromosomal transgenes of 1-20 copies. Then we will screen through Southern analysis for lines that are single copy and integrated and work (Rose A., 2008). Such an assay would be much more laborious resulting in many lines screen therefore; working with Mos transoposon is our first choice.