Mutation in pre-mRNA sequence

Mutation in pre-mRNA sequence

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Has there any mutations been recorded which cause harmful effects due to change in the part of pre-mRNA responsible for proper m-RNA splicing ?

Yes, you can find mutations in the genomic DNA which affect splice acceptor sites. Wikipedia lists the following outcome:

  • Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron.
  • Mutation of a splice site reducing specificity. May result in variation in the splice location, causing insertion or deletion of amino acids, or most likely, a disruption of the reading frame.
  • Displacement of a splice site, leading to inclusion or exclusion of more RNA than expected, resulting in longer or shorter exons.

The article lists two interesting papers:

Lim KH, Ferraris L, Filloux ME, Raphael BJ, Fairbrother WG (2011). "Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes". Proc. Natl. Acad. Sci. U.S.A. 108 (27) 11093-11098.

Ward AJ, Cooper TA (2010). "The pathobiology of splicing". J. Pathol. 220 (2) 152-163.

Both papers are open access.

Assessment of the functional impact on the pre-mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha-glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre-messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.-32-13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.

Keywords: RNA splicing mutations antisense oligonucleotides glycogenosis type 2 mRNA splicing pompe disease.

Splicing mutations in human genetic disorders: examples, detection, and confirmation

Precise pre-mRNA splicing, essential for appropriate protein translation, depends on the presence of consensus "cis" sequences that define exon-intron boundaries and regulatory sequences recognized by splicing machinery. Point mutations at these consensus sequences can cause improper exon and intron recognition and may result in the formation of an aberrant transcript of the mutated gene. The splicing mutation may occur in both introns and exons and disrupt existing splice sites or splicing regulatory sequences (intronic and exonic splicing silencers and enhancers), create new ones, or activate the cryptic ones. Usually such mutations result in errors during the splicing process and may lead to improper intron removal and thus cause alterations of the open reading frame. Recent research has underlined the abundance and importance of splicing mutations in the etiology of inherited diseases. The application of modern techniques allowed to identify synonymous and nonsynonymous variants as well as deep intronic mutations that affected pre-mRNA splicing. The bioinformatic algorithms can be applied as a tool to assess the possible effect of the identified changes. However, it should be underlined that the results of such tests are only predictive, and the exact effect of the specific mutation should be verified in functional studies. This article summarizes the current knowledge about the "splicing mutations" and methods that help to identify such changes in clinical diagnosis.

Keywords: Pre-mRNA splicing Spliceosome Splicing enhancers and silencers Splicing mutation.

Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Arabidopsis intron mutations and pre-mRNA splicing

Arabidopsis intron mutants provide and will continue to provide a valuable source of information on in vivo plant intron splicing. All of the characterized mutants discussed here contain base substitutions in either the 5' splice site :GU or 3' splice site AG: dinucleotides or broader splice site consensus sequences. Many of these mutations lead to the activation of cryptic splice sites, usually upstream or downstream of the authentic 5' and 3' splice sites respectively, often with reduced efficiency. This splicing behaviour is in agreement with detailed splicing analyses of test plant introns. However, some of the Arabidopsis mutations lead to more complex splicing patterns often involving exon skipping. These mutations illustrate the complexity of the splicing reaction (where the final splicing event reflects the characteristics such as splice site sequence, intron size and composition, and their interactions with spliceosomal components) and how single nucleotide mutations can affect the strength and balance of interactions to alter splicing patterns. The splicing patterns observed in the Arabidopsis mutants parallel those seen in mutations causing some human genetic disorders underlining the emerging similarities in mechanisms of splice site selection and intron/exon definition between plant and vertebrate systems. Analysis of the Arabidopsis intron mutations exhibiting complex splicing patterns will help to address fundamental questions in plant splicing, such as splice site selection and exon scanning. This information will be important in understanding the mechanisms by which gene expression is regulated post-transcriptionally in the ever-increasing number of alternatively spliced plant gene systems.


Translocations are the transfer of a piece of one chromosome to a nonhomologous chromosome. Translocations are often reciprocal that is, the two nonhomologues swap segments.

Figure 10.1.6 Translocations

Translocations can alter the phenotype is several ways:

  • the break may occur within a gene destroying its function
  • translocated genes may come under the influence of different promoters and enhancers so that their expression is altered. The t(814) translocation in Burkitt's lymphoma (figure) is an example.
  • the breakpoint may occur within a gene creating a hybrid gene. This may be transcribed and translated into a protein with an N-terminal of one normal cell protein coupled to the C-terminal of another. The Philadelphia chromosome found so often in the leukemic cells of patients with chronic myelogenous leukemia (CML) is the result of a translocation which produces a compound gene (bcr-abl).

76 RNA Processing in Eukaryotes

By the end of this section, you will be able to do the following:

  • Describe the different steps in RNA processing
  • Understand the significance of exons, introns, and splicing for mRNAs
  • Explain how tRNAs and rRNAs are processed

After transcription, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated. Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein-synthesis machinery.

MRNA Processing

The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated. Eukaryotic protein-coding sequences are not continuous, as they are in prokaryotes. The coding sequences (exons) are interrupted by noncoding introns, which must be removed to make a translatable mRNA. The additional steps involved in eukaryotic mRNA maturation also create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.

Pre-mRNAs are first coated in RNA-stabilizing proteins these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of the introns ((Figure)). In rare cases, the mRNA transcript can be “edited” after it is transcribed.

The trypanosomes are a group of protozoa that include the pathogen Trypanosoma brucei, which causes nagana in cattle and sleeping sickness in humans throughout great areas of Africa ((Figure)). The trypanosome is carried by biting flies in the genus Glossina (commonly called tsetse flies). Trypanosomes, and virtually all other eukaryotes, have organelles called mitochondria that supply the cell with chemical energy. Mitochondria are organelles that express their own DNA and are believed to be the remnants of a symbiotic relationship between a eukaryote and an engulfed prokaryote. The mitochondrial DNA of trypanosomes exhibit an interesting exception to the central dogma: their pre-mRNAs do not have the correct information to specify a functional protein. Usually, this is because the mRNA is missing several U nucleotides. The cell performs an additional RNA processing step called RNA editing to remedy this.

Other genes in the mitochondrial genome encode 40- to 80-nucleotide guide RNAs. One or more of these molecules interacts by complementary base pairing with some of the nucleotides in the pre-mRNA transcript. However, the guide RNA has more A nucleotides than the pre-mRNA has U nucleotides with which to bind. In these regions, the guide RNA loops out. The 3′ ends of guide RNAs have a long poly-U tail, and these U bases are inserted in regions of the pre-mRNA transcript at which the guide RNAs are looped. This process is entirely mediated by RNA molecules. That is, guide RNAs—rather than proteins—serve as the catalysts in RNA editing.

RNA editing is not just a phenomenon of trypanosomes. In the mitochondria of some plants, almost all pre-mRNAs are edited. RNA editing has also been identified in mammals such as rats, rabbits, and even humans. What could be the evolutionary reason for this additional step in pre-mRNA processing? One possibility is that the mitochondria, being remnants of ancient prokaryotes, have an equally ancient RNA-based method for regulating gene expression. In support of this hypothesis, edits made to pre-mRNAs differ depending on cellular conditions. Although speculative, the process of RNA editing may be a holdover from a primordial time when RNA molecules, instead of proteins, were responsible for catalyzing reactions.

5′ Capping

While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5′ end of the growing transcript by a phosphate linkage. This functional group protects the nascent mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.

3′ Poly-A Tail

Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail . This modification further protects the pre-mRNA from degradation and is also the binding site for a protein necessary for exporting the processed mRNA to the cytoplasm.

Pre-mRNA Splicing

Eukaryotic genes are composed of exons , which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (int-ron denotes their intervening role), which may be involved in gene regulation but are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.

The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns. These regions may correspond to regulatory sequences however, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout the course of evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.

All of a pre-mRNA’s introns must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing ((Figure)). Introns are removed and degraded while the pre-mRNA is still in the nucleus. Splicing occurs by a sequence-specific mechanism that ensures introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. Although the intron itself is noncoding, the beginning and end of each intron is marked with specific nucleotides: GU at the 5′ end and AG at the 3′ end of the intron. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.

Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors? Think of different possible outcomes if splicing errors occur.

<!– <link window=”new” target-id=”fig-ch15_04_02″ document=””/>Mutations in the spliceosome recognition sequence at each end of the intron, or in the proteins and RNAs that make up the spliceosome, may impair splicing. Mutations may also add new spliceosome recognition sites. Splicing errors could lead to introns being retained in spliced RNA, exons being excised, or changes in the location of the splice site. –>

Note that more than 70 individual introns can be present, and each has to undergo the process of splicing—in addition to 5′ capping and the addition of a poly-A tail—just to generate a single, translatable mRNA molecule.

See how introns are removed during RNA splicing at this website.

Processing of tRNAs and rRNAs

The tRNAs and rRNAs are structural molecules that have roles in protein synthesis however, these RNAs are not themselves translated. Pre-rRNAs are transcribed, processed, and assembled into ribosomes in the nucleolus. Pre-tRNAs are transcribed and processed in the nucleus and then released into the cytoplasm where they are linked to free amino acids for protein synthesis.

Most of the tRNAs and rRNAs in eukaryotes and prokaryotes are first transcribed as a long precursor molecule that spans multiple rRNAs or tRNAs. Enzymes then cleave the precursors into subunits corresponding to each structural RNA. Some of the bases of pre-rRNAs are methylated that is, a –CH3 methyl functional group is added for stability. Pre-tRNA molecules also undergo methylation. As with pre-mRNAs, subunit excision occurs in eukaryotic pre-RNAs destined to become tRNAs or rRNAs.

Mature rRNAs make up approximately 50 percent of each ribosome. Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic or binding activities. Mature tRNAs take on a three-dimensional structure through local regions of base pairing stabilized by intramolecular hydrogen bonding. The tRNA folds to position the amino acid binding site at one end and the anticodon at the other end ((Figure)). The anticodon is a three-nucleotide sequence in a tRNA that interacts with an mRNA codon through complementary base pairing.

Section Summary

Eukaryotic pre-mRNAs are modified with a 5′ methylguanosine cap and a poly-A tail. These structures protect the mature mRNA from degradation and help export it from the nucleus. Pre-mRNAs also undergo splicing, in which introns are removed and exons are reconnected with single-nucleotide accuracy. Only finished mRNAs that have undergone 5′ capping, 3′ polyadenylation, and intron splicing are exported from the nucleus to the cytoplasm. Pre-rRNAs and pre-tRNAs may be processed by intramolecular cleavage, splicing, methylation, and chemical conversion of nucleotides. Rarely, RNA editing is also performed to insert missing bases after an mRNA has been synthesized.

Visual Connection Questions

(Figure) Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors? Think of different possible outcomes if splicing errors occur.

(Figure) Mutations in the spliceosome recognition sequence at each end of the intron, or in the proteins and RNAs that make up the spliceosome, may impair splicing. Mutations may also add new spliceosome recognition sites. Splicing errors could lead to introns being retained in spliced RNA, exons being excised, or changes in the location of the splice site.

Review Questions

Which pre-mRNA processing step is important for initiating translation?

What processing step enhances the stability of pre-tRNAs and pre-rRNAs?

A scientist identifies a pre-mRNA with the following structure.

What is the predicted size of the corresponding mature mRNA in base pairs (bp), excluding the 5’ cap and 3’ poly-A tail?

Critical Thinking Questions

Chronic lymphocytic leukemia patients often harbor nonsense mutations in their spliceosome machinery. Describe how this mutation of the spliceosome would change the final location and sequence of a pre-mRNA.

Nonsense spliceosome mutations would eliminate the splicing step of mRNA processing, so the mature mRNAs would retain their introns and be perfectly complementary to the entire DNA template sequence. However, the mRNAs would still undergo addition of the 5’ cap and poly-A tail, and therefore each has the potential to be exported to the cytoplasm for translation.


Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm

We designed a strategy to identify splicing factors that act by preventing pre-mRNA transport into the cytoplasm. A yeast synthetic intron was inserted into a lacZ gene so that only the pre-mRNA could be translated to produce beta-galactosidase activity. Deletion of either of the 5' splice junction sequence GUAUGU and the branchpoint sequence UACUAAC resulted in a dramatic increase in pre-mRNA translation, indicating its cytoplasmic localization. In rna6 and rna9 mutant strains assayed at the nonpermissive temperature, splicing inhibition occurred simultaneously with a large increase in pre-mRNA translation. Similarly, a point mutation in U1 snRNA decreased splicing efficiency and increased pre-mRNA translation. From these results, we conclude that early acting factors, probably including U1 snRNA, and the RNA6 and RNA9 gene products, interact in vivo with the 5' splice junction and the branchpoint sequence to commit the pre-mRNA to the splicing pathway, thereby preventing its transport to the cytoplasm.

Mutations in the RNA binding domain of stem-loop binding protein define separable requirements for RNA binding and for histone pre-mRNA processing

Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3' untranslated region of histone pre-mRNAs and facilitate 3' end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial loss of SLBP binding activity. We also identify residues in the RBD which do not contribute to binding to the stem-loop RNA but instead are required for efficient recruitment of U7 snRNP to histone pre-mRNA. Recruitment of the U7 snRNP to the pre-mRNA also depends on the 20-amino-acid region located immediately downstream of the RBD. A critical region of the RBD contains the sequence YDRY. The tyrosines are required for RNA binding, and the DR dipeptide is essential for processing but not for RNA binding. It is likely that the RBD of SLBP interacts directly with both the stem-loop RNA and other processing factor(s), most likely the U7 snRNP, to facilitate histone pre-mRNA processing.


Conserved amino acids within the…

Conserved amino acids within the RBD of the SLBP. (A) Schematic of the…

The conserved amino acids in…

The conserved amino acids in the RBD are required for efficient binding of…

High-affinity stem-loop binding is required…

High-affinity stem-loop binding is required for efficient processing of histone H1t pre-mRNA but…

Amino acids conserved in the…

Amino acids conserved in the RBD of human SLBP and xSLBP1 but not…

Substitution of nine amino acids…

Substitution of nine amino acids within the RBD of human SLBP abolishes processing…

Activity of 9aaR and 20aaC…

Activity of 9aaR and 20aaC mutant SLBPs in 3′ end processing is substrate…

Mutant proteins 9aaR and 20aaC…

Mutant proteins 9aaR and 20aaC inhibit H1t pre-mRNA processing when added to a…

9aaR and 20aaC mutant SLBPs…

9aaR and 20aaC mutant SLBPs have significantly reduced ability to recruit the U7…

Watch the video: Splicing (October 2022).