Splicing of Intronic Sequences

The intervening sequences or the introns do not code for any proteins. However, they are present in the gene separating the coding sequences from each other. They were discovered recently, a few decades back. Till now we only know a few types of intronic sequences and their locations. Some belong to nuclear pre-RNAs whereas others belong to organelle pre-RNAs. Complete maturation of RNA is tough to achieve without the removal of intronic sequences. Hence, without splicing of introns, RNA does not get translated into a protein. The pre-mRNA consists of an alternative arrangement of introns and exons. After a particular exonic sequence, an intronic sequence starts, followed by exons and introns again. Also, a mRNA consists of many introns with lengthy sequences covering the majority of the mRNA transcript. Thus, they take up a considerable length of the transcript, and thus they get spliced or excised. The exons, on the other hand, have specific sequences coding for amino acids. Hence they need to be ligated together.

There are total eight types of introns known till now. The eukaryotic nuclear pre-RNAs consist of the GU-AG introns and the AU-AC introns. A specific class of introns known as a group I introns occur in the eukaryotic nuclear pre-rRNA as well as organelle RNAs. The group II and group III introns occur only in the organelle RNAs. Next type of introns is known as Twintrons. They are a simple type of introns consisting of one intron embedded in the other intron. Complex Twintrons consist of multiple embedded introns. They are also present in the organelle RNAs. Nuclear pre-tRNAs consist of pre-tRNA introns. The above types of introns belong to eukaryotic RNAs. However, the group I and group II introns also belong to prokaryotic RNAs. There are only a few pieces of evidence regarding the archeal introns. The spliceosomes help in carrying out this important process. They consist of pre-mRNAs bound to small nuclear ribonucleoprotein particles known as snRNPs or snurps. They are small nuclear RNAs with protein complexes. Five principal snRNAs include U1, U2, U4, U5, and U6, and are associated with proteins to form snRNPs.

Events before the initiation of splicing:

A complex known as the commitment complex initiates the splicing activity. It comprises U1SnRNP. The U1 binds to 5’ splice site by RNA-RNA base pairing. Certain proteins act as binding factors. The examples include SF1, U2AF35, and U2AF65. These binding proteins help to make protein-RNA contacts with the branch site, the polypyrimidine tract and the 3’ splice site respectively. Another important complex known as pre-spliceosome complex consists of the above commitment complex along with another snRNP known as U2-snRNP. This snurp attaches to the branch site. The synergy of U1 snRNP and U2 snRNP brings the 5’ splice site near the branch point. Hence, the commitment complex and the pre-spliceosome complex assist the spliceosome. A spliceosome is a protein-RNA complex that removes the introns. A spliceosome complex forms when U4/U6 snurps and the U5-snRNP attach to the pre-spliceosome complex. It leads to the movement of 3’ splice site closer to the 5’ site and the branch point. Now, the two esterification reactions occur. The reaction gets catalyzed by the U6-snRNPs. An important group of proteins known as splicing factor proteins or SR proteins assists in the splice site selection. The word “SR” indicates a C-terminal domain containing serine and arginine.

Steps involved in splicing

1.     U1 snRNP binds to 5’ splice junction of the intron. It occurs due to the base pairing of the U1 snRNA in the snRNP at the 5’ splice junction.

2.     U2 snRNP binds to a sequence known as branch point sequence located at the 3’ splice site junction.

3.   U4/U6 snRNP and a U5 snRNP interact with each other and bind to the U1 and U2 snRNPs resulting in looping of the intron. Hence, they bring two ends closer.

4.     The U4 snRNP gets dissociated.

5.     Then forms the active spliceosome.

6.    The snRNP cleaves the introns from the first exon at the 5’ splice junction. The 5’ end of the intron gets bound with an adenine nucleotide in the branch point sequence. Hence, the structure formed out of this activity is known as a lariat.

7.   The intron gets excised by cleavage at the 3’ junction. Thus, the first and the second exons get ligated together.

Image: Splicing procedure

Splicing studies in Tetrahymena

The process of splicing in the Tetrahymena is known as self-splicing. It occurs in the pre-rRNA. Following are the steps:

1.     The cleavage occurs at the 5’ splice junction accompanied by an addition of G- nucleotide to the 5’ end of the intron.

2.     The cleavage at the 3’ splice junction releases the intron. Then the two exons get spliced.

3.     Next, the intron starts circularizing by a 3’ nucleotide bonding to an internal nucleotide, thereby producing a lariat.

4.     The last step involves cleavage of the lariat to produce a circular RNA and a linear molecule.

Types of the aberrant splicing:

Sometimes aberrations happen even in simple processes such as splicing. There are two forms of aberrant splicing. The first one is known as an exon skipping. In this, the aberrant splicing results in loss of one exon from the mRNA. The other form of aberrant splicing involves selection of a cryptic splice site. In this type of selection, there is a loss of an important part of an exon from the mRNA. It alternatively results in the selection of an intron to be retained.

Alternative splicing

It is a crucial finding in the field of genomics. Alternative splicing enables a single transcript processed in a related but a different mRNA. It leads to the synthesis of a variety of proteins.

1.     Alternative splicing in Drosophila:

It occurs during sex determination and gene regulation. The beginning of the cascade involves sex-specific alternative splicing of the Sxl pre-mRNA. A full length functional Sxl protein gets synthesized in females because the third exon gets skipped in the females, unlike males. The Sxl blocks the 3’ splice site in the first intron TRA pre-mRNA. It encodes a functional TRA protein. In males, the fourth exon of DSX pre-mRNA gets skipped. It encodes a male-specific protein known as DSX protein. The mechanism for encoding female specific protein involves a different strategy.

2.     Alternative splicing in humans:

A gene in humans known as an SLO gene comprises 35 exons in all. It encodes for a membrane protein regulating the entry and exit of the potassium ions in and out of the cells. There is an involvement of the total of eight exons. These genes are active in the inner ear. They determine the auditory properties of the hair cells on the basilar membrane of the cochlea. About 40-60% of human genes have alternative splice forms. Hence, alternative splicing is very important in the human genome and its functional complexity.

Trans-splicing in C. elegans:

The exons present in different RNA molecules get spliced in the trans-splicing process. C. elegans consists of SLRNA. It is known as a spliced leader RNA. It is almost 100 nucleotides in length. Here, a fork forms instead of a lariat. Some genes in C. elegans get transcribed in pairs from a single promoter.

Eukaryotic pre-rRNAs and pre-tRNA splicing:

Eukaryotes consist of autocatalytic pre-rRNA intron sequences. These introns belong to the group I family of introns and found in mitochondrial and chloroplast genomes. They occur in pre-rRNA and pre-mRNA. The first reaction involves a free nucleoside. The second transesterification involves attack of the 3’-OH to the phosphodiester bond at 3’ splice site causing cleavage and release of an exon. For releasing the introns from the pre-tRNA, introns do not involve trans-esterification reaction. An endonuclease cuts the two splice sites. 

References:
[1] Molecular Biology of the Cell, Bruce Alberts
[2] Molecular Biology of RNA, David Elliott, Michael Ladomery
[3] IGenetics, Peter Russell, second edition
[4] Gene Regulation: A Eukaryotic Perspective, David S. Latchman

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