Why is cdna shorter than mrna

These nucleotides are coupled to a deoxyribose sugar and are able to bind to other deoxyribose sugars via phosphate linkages to form long chains, some of which can be well over ,, molecules long.

Since each deoxyribose in a DNA chain is coupled to one of the four nitrogenous bases G, A, T, or C , these long chains can carry information. Codons are used to call for specific amino acids to be bonded together to form proteins. For instance the codon adenosine-adenosine-guanosine AAG calls for the amino acid lysine lys to be incorporated into a protein molecule. The codon AGG calls for the amino acid arginine arg. There are also codons that, under the right circumstances, call for a protein to begin to be formed start codons , or for a protein chain to be finished stop codons.

As you can see from this simple example, DNA can carry a massive amount of information. Figure 1: Adenine binds to thymine; guanine binds to cytosine. A gene is a set of codons that specify a specific protein chain, along with the associated start and stop codons. In nature, the process for information to be passed on from DNA can occur through either replication or gene expression. There are some important factors to note:. Initially, it was observed that gDNA was always read and transcribed into mRNA, which guided protein formation and then was disposed.

Calling it that challenged scientists to find exceptions to this rule. Virologists eventually did find one such exception. It should be noted that prokaryotes are not capable of splicing out introns. Exons are a necessary part of the coding system, being retained after introns are spliced out. This is displayed in Figure 3. Exitrons are introns that are not spliced out, despite containing no coding sequences.

When scientists use viral enzymes to make cDNA from RNA isolated from the cells and tissues that they are studying, it does not contain introns due to being spliced out in mRNA. IV , FIX variant missing the CDS completely for the activation peptide, nearly full-length of light chain and 41 aa long domain in the heavy chain a. Here, we inferred four different motifs for acceptor and donor sites based on sequencing data.

In the intron-exon junction, the acceptor site, the motif CAG is conserved in 3 out of 4 cases, but the adjacent two nucleotides do not follow any trend. Four different splicing motifs were identified after sequencing FIX variants. Left side shows donor site and right side shows acceptor site.

Consensus genomic sequence motifs of donor b and acceptor splice sites c extracted from all 32, P. The probability and height of individual letters correspond to base frequencies at each position. To analyze whether donor and acceptor sites and their neighboring nucleotides are conserved in P. The full list of splice sites is compiled in Supplementary Tables 2 and 3. Subsequently, we analyzed the FIX CDS with a moss splice-site prediction tool 46 , but it largely failed to predict the experimentally verified splicing motifs.

This might be due to the fact that this tool has been developed several years ago using only donor and acceptor sites 46 and has not been updated since then. The analyses at RNA level suggested that P. To validate this inference, the culture supernatants of transiently transfected cells were precipitated and analyzed with a polyclonal anti-FIX antibody via immunoblot after reduction and alkylation Fig.

In addition, we used the other half of the supernatants for mass-spectrometric MS determination of FIX-variant peptide sequences. For the MS-database search, protein models for FIX variants derived from the sequenced splice variants were employed. Culture supernatant of non-transfected cells was used as negative control Neg.

Amino acids marked in yellow were identified in the MS analysis. This result confirmed the presence of a predicted FIX variant caused by heterosplicing on an additional level. Unedited blot showing the full lanes can be found in Supplementary Fig.

In the first one, aspFIX, the codons neighboring the detected splice junctions were modified. On day 14, the cells were collected and RNA was isolated. The donor and acceptor sites of the shorter variant were identified Fig. Moreover, splicing would cause a frameshift mutation resulting in early stop codons.

Despite the fact that splicing and translation take place in different cellular compartments we next performed a complete codon optimization of the FIX CDS in the aspFIX construct according to previous studies 49 , 50 resulting in the optiFIX sequence with an overall increased GC content.

Although translatability of a particular RNA via codon usage is not expected to regulate whether it can be spliced or not, we aimed to change the recognition of parts of human CDS regions as intronic regions by the moss spliceosome machinery. This was used to transiently transfect moss cells.

The presence of exclusively the full-length CDS was confirmed on protein level via immunoblot analysis Fig. An unedited blot showing the full lanes can be found in Supplementary Fig. The left side shows the donor site and the right side the acceptor site. Next, we compared the codon usage bias in P. On the other hand, this codon is underrepresented in N. The synonymous codon usage bias in P. Subsequently, we transiently transfected moss protoplasts with these constructs, and performed confocal laser scanning microscopy CLSM on day 3 after transformation.

A visual analysis of the images revealed that the FIX-Citrine signal shows a diffuse localization pattern, which might be due to intracellular mislocalization of aberrant protein isoforms Fig. In contrast, optiFIX-Citrine exhibited a complex network-like organization, indicating a proper localization of the protein in the ER Fig.

The source images Z-stacks for Fig. The slice with the brightest mean intensity was extracted from a Z-stack and its voxel intensity range was adjusted to 0—36 for illustration. In order to automatize the process of P. In addition, physCO checks the input for consistent lengths, valid start, and stop codons, and internal, premature stop codons and raises warnings in case it detects irregularities. In order to test whether heterosplicing is a common phenomenon in P.

These changes affected of codons, and increased the GC content from To investigate the effects of these alterations in live moss cells, we cloned two fusion constructs, FH-Citrine and optiFH-Citrine.

Subsequently, we transiently transfected moss protoplasts with these constructs, and performed CLSM on day 3 after transformation. Analysis on FH showed that heterosplicing is not an effect specific for FIX, but a general phenomenon in moss. Therefore, transgene sequences have to be modified using physCO prior to transformation for proper transcription and translation. Heterologous gene expression is of fundamental importance for basic biology and for industrial production.

In order to optimize transgene transcription and translation as well as transcript stability, many factors have to be taken into account. Engineering of upstream and downstream regulatory sequences, replacing original signal peptides with host-suitable ones and optimizing codons according to host codon-usage pattern can be used to improve protein production. Generation of these protein isoforms can affect fundamental characteristics of the protein such as function, activity, intracellular localization, interaction with other molecules, and stability.

Moreover, splicing can create a transcript with an early stop codon, which may be degraded by NMD. Thus, it can be hypothesized that modulation of splicing, a neglected point in transgene expression, can improve protein production.

According to RT-PCR analyses, this phenomenon occurred not only in juvenile protonema, but also in adult moss plants. We sequenced these mRNAs and surprisingly found that reading frames had not changed in the four smaller variants in comparison to the mature full-length transcript. This may have happened by a pure coincidence. Alternatively, we may not have been able to detect such putative minor transcripts because they were either too low abundant for RT-PCR, or they have been eliminated by NMD.

The outcome of this heterosplicing was detectable on four levels, by RT-PCR from mRNAs, immunoblot, mass spectrometry of the protein isoforms, and in vivo by confocal imaging showing deviant localization of Citrine reporter fusions.

Our observations revealed that heterosplicing of the human FIX mRNA in moss is consistent across different tissues and not dependent on developmental stages.

Furthermore, the consistency of heterosplicing in episomally transiently FIX-expressing cells indicates that heterosplicing of the FIX mRNA in transgenic lines was neither due to illegitimate or partial integration of the construct into the moss genome nor a locus-specific effect.

It caused the generation of various stable FIX mRNA variants and protein isoforms in addition to the complete transcript and protein. Therefore, a possible occurrence of heterosplicing should be analyzed on RNA level, even if a full-length protein can be detected in heterologous cells. In addition, we identified consensus motifs of donor and acceptor splice sites in the latest P.

This revealed that P. Previous work by Marquez et al. Moreover, plant introns are different from animal introns in terms of UA- or U-richness, which is crucial for splicing efficiency Due to these characteristic differences, it is likely that the GC content can affect the characteristics of splice site recognition The GC content is correlated with many features including gene density 56 , intron length 57 , meiotic recombination 58 , and gene expression It varies among species, and even along chromosomes It was shown before that the average GC content in P.

A closer comparison of the aspFIX and optiFIX constructs revealed that there was no change in the donor and acceptor sites of the newly emerged cryptic intron. There was, however, an increase of the GC content within the cryptic intron sequence. As a result, a nearly fold increase in protein amounts was achieved in transiently transfected moss protoplasts in vivo.

As heterosplicing did not cause detectable frameshift mutations, fluorescence signals obtained from the FIX-Citrine construct were the sum of all FIX protein isoforms, full-length and fragmentary, fused to Citrine. Based on this inference and proteins, which were harvested from transient transformation with the FIX construct and detected on the immunoblot Fig.

Two mechanisms, namely boosted translation rates and the prevention of mRNA degradation via NMD, may contribute to this result.

In addition, the protein from the optiFIX construct was solely detectable in the ER, the compartment of choice for correct posttranslational modifications and subsequent secretion of the protein. A similar phenomenon was observed in the expression of another transgene in Physcomitrella. One example of heterosplicing and its prevention by our methodology is the expression of human complement FH.

FH is an important regulator of the alternative pathway in the human complement system. Currently, a recombinant FH is not available on the market, although it has potential as a biopharmaceutical in the treatment of severe human diseases like atypical hemolytic uremic syndrome aHUS , age-related macular degeneration AMD or C3 glomerulopathies.

The recombinant production of FH, devoid of plant-specific N-linked sugar residues, in moss resulted in a range of promising biological activities Moreover, moss-derived FH is currently being examined for use in Covid treatment www.

A visual analysis of the images acquired for FH and optiFH from transiently transfected moss cells revealed that while FH-Citrine signal intensity is not detectable, optiFH-Citrine signal intensity is high Supplementary Fig. Thus, an optimization of the currently used CDS of human FH in plants is advantageous for plant-based production. These findings suggest that the splicing of heterologous mRNAs should be taken into account on a routine basis.

In addition, our analysis of synonymous codon usage bias suggests that our methodology to prevent heterosplicing can be directly implemented in organisms, which follow similar codon usage patterns as P. Our methodology might still be vital in organisms that have different codon usage patterns, such as the species of the genus Nicotiana , e. Mutating donor and acceptor sites and their neighboring nucleotides together with replacing codons ending with A or T to G or C may prevent the generation of heterosplice variants.

Why should splice site mutation and codon optimization be employed at the same time rather than codon optimization alone? Although differences between GC-rich and GC-poor genes were not reported in moss, analyses of various plant species can explain why codon optimization might not be sufficient.

Hence, increasing the GC content of cryptic introns to a certain level without mutating splice site motifs might not be sufficient to prevent aberrant mRNA processing.

In general, the analysis of spliced transcripts is beneficial for at least five reasons. Prevention of heterosplice variants eventually will improve recombinant protein production and decrease downstream processing costs significantly. Hence, plant-based systems can become an alternative to traditional production platforms.

Moreover, it may have implications for basic biology as well, because here the use of heterologous reporter constructs is of vital importance. For these purposes, we developed physCO, an automated P. Moreover, our analysis of codon usage patterns indicates that this tool can also be used for insect S. Besides that, we cannot exclude the possibility, that the phenomenon of mRNA splicing, here described as heterosplicing, is a novel gene regulatory mechanism occurring in eukaryotes in general.

For codon optimization, two sources, codon usage bias calculated by Hiss et al. Underrepresented codons were replaced with the overrepresented alternative codons Table 2 via physCO. See Supplementary Fig. It was used in transient expression assays. It was cloned into the same vector as FH. In addition to the generation of transgenic lines, mossFIX was produced transiently by an upscaled transformation protocol based on the proportion of 0.

Cells transiently expressing mossFIX were grown in special regeneration medium 7. After selection on solidified Knop medium, transgenic lines were cultured under standard conditions in liquid Knop medium Following the hygromycin selection process, stable lines were characterized by the presence of the FIX transcript.

For analysis of gametophores, 5—6 gametophores were collected and RNA isolation was performed as described above. The PCR products were examined by standard agarose gel electrophoresis with visualization of DNA by ethidium bromide fluorescence. Following the transformation, cells were grown in regeneration medium for 2 weeks Transiently transfected cells nearly 3.

Sequencing was done as described above. The most recent release of the Physcomitrella v3. Consensus sequence logos of all extracted donor and acceptor sites were created using R and the package ggseqlogo 69 , Synonymous codon usage biases in P. The voxel sizes were 0. The pinhole was adjusted to 1 Airy Unit The image processing consists of the following steps: i denoising, ii edge enhancement, iii Richardson—Lucy restoration, iv local intensity equalization, v segmentation.

In the first step, a median filter with a window of 3,3,3 was applied to the images in order to remove the salt-and-pepper noise.

In the second step, the denoised images were subjected to an unsharp-mask operation 71 , 72 , which involves blurring the image with a Gaussian filter, subtracting the blurred image from the input image, and adding the resulting difference back to the input image, a process that sharpens the edges in the images.

The edge-enhanced images were then subjected to the Richardson—Lucy restoration algorithm, as implemented in the scikit-image package 73 for three iterations, assuming an averaging filter in a window of 3,5,5 as point spread function here the aim was to smooth the image without losing the thin structures, rather than a true deblurring. The code for the Richardson—Lucy algorithm was adapted from scikit-image package 73 using Python programming language.

Subsequently, a spatial intensity equalization step was implemented on the images using a home-built algorithm. In short, each image was divided into boxes with dimensions of 7,7,7 voxels. The voxel values within these boxes were rescaled by multiplying the values in each box with weights that are calculated based on the skewness value corresponding to the same box.

This step corrected for the intensity gradients in the image in order to minimize the loss of some low-intensity foreground voxels.

The code for the adaptive Otsu thresholding algorithm was developed based on the Otsu threshold function of the scikit-image package 73 and the Numba package The adaptive thresholding operation yielded the binary masks that specified the foreground voxels, which in turn were used to calculate the mean voxel intensity for each original image.

All six FIX images were processed and quantified in a single run of a Python script by using the exact same parameters to avoid any possible bias. The code used for image processing and quantification is deposited on from www. The procedure is shown in Supplementary Fig.

Proteins from the culture supernatant were precipitated as described For Western blot analyses, 7. Afterwards, the membrane was washed three times with TBS containing 0. MS analyses were performed using a binary solvent system consisting of 0. Samples were washed and concentrated on a C18 precolumn with 0. MS parameters were as follows: spray voltage 1. Raw data were analyzed using Mascot Distiller V2. The peptide mass tolerance was set to 8 ppm and the fragment mass tolerance was set to 0.

A total of two missed cleavages was allowed as well as semitryptic peptides. Search results were loaded into Scaffold4 software Version 4. The sample size was decided based on prior experience.

Further information on research design is available in the Nature Research Reporting Summary linked to this article. All data generated or analyzed during this study are included in this article and its supplementary information. Microscopy images can be found in Supplementary Data 1 and 6.

An interactive version of physCO as well as the code used for the image processing and quantification are available at www. Sakharkar, M. Distributions of exons and introns in the human genome. Silico Biol. CAS Google Scholar. Georgomanolis, T. Cutting a long intron short: recursive splicing and its implications. Rogozin, I. Origin and evolution of spliceosomal introns. Direct 7 , 11 Papasaikas, P. The spliceosome: the ultimate RNA chaperone and sculptor.

Trends Biochem. Berget, S. Exon recognition in vertebrate splicing. Goldstrohm, A. Co-transcriptional splicing of pre-messenger RNAs: Considerations for the mechanism of alternative splicing. Gene , 31—47 Dredge, B. The splice of life: alternative splicing and neurological disease.

Shai, O. Inferring global levels of alternative splicing isoforms using a generative model of microarray data. Bioinformatics 22 , — Nissim-Rafinia, M. Splicing regulation as a potential genetic modifier. Trends Genet. Yu, J. Genome Res. Nilsen, T. Expansion of the eukaryotic proteome by alternative splicing. Nature , — Sanchez, L. Sex-determining mechanisms in insects. Shang, X. Alternative splicing in plant genes: a means of regulating the environmental fitness of plants.

Deckert, J. Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions. Amit, M. Differential GC content between exons and introns establishes distinct strategies of splice-site recognition. Cell Rep. Carmel, I. RNA 10 , — Chiara, M. EMBO J. Hall, S. Conserved sequences in a class of rare eukaryotic nuclear introns with non-consensus splice sites.

Computational screen for spliceosomal RNA genes aids in defining the phylogenetic distribution of major and minor spliceosomal components. Nucleic Acids Res. Syed, N. Alternative splicing in plants - coming of age. Trends Plant Sci. Pan, Q. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Zimmer, A. Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions.

BMC Genom. Lloyd, J. The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens. Melo, J. Current challenges in studying alternative splicing in plants: The case of Physcomitrella patens SR Proteins. Plant Sci. Schellenberg, M. Pre-mRNA splicing: a complex picture in higher definition. Maniatis, T. An extensive network of coupling among gene expression machines.

Reed, R. Initial splice-site recognition and pairing during pre-mRNA splicing. Ram, O. SR proteins: a foot on the exon before the transition from intron to exon definition. Gelfman, S.

Changes in exon-intron structure during vertebrate evolution affect the splicing pattern of exons. Haseloff, J. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Natl Acad. USA 94 , — Diehn S. Vol 18 ed. Setlow J. Springer, Boston, MA, Plant Mol.

PubMed Article Google Scholar. Top, O. Critical evaluation of strategies for the production of blood coagulation factors in plant-based systems. Kurachi, K. Biology of factor IX. Chen, C. Economic burden of illness among persons with hemophilia b from HUGS Vb: examining the association of severity and treatment regimens with costs and annual bleed rates. Value Health 20 , — Decker, E. Mosses in biotechnology.