Deep Insight of Intron Splicing Mechanism
Author: Sachidanand Tiwari

INTRODUCTION

Introns are intervening sequences that are removed from primary transcripts by the process of RNA splicing, which links together the flanking pieces (exons) to generate functional mature RNAs. Efficient removal of introns, with single nucleotide precision is essential for eukaryotic cells to produce the correct complement of mRNA. Introns are extremely common within the nuclear genome of higher vertebrates like mammals where protein-coding genes almost always contain multiple introns, while introns are rare within the nuclear genes of some eukaryotic microorganisms. On basis of examination of intron structure by DNA sequence analysis, together with genetic and biochemical analysis of splicing reactions, introns are classified in three classes.

1. Group I and group II introns

Group I and group II introns are found in genes encoding proteins messenger RNA, transfer RNA and ribosomal RNA in a very wide range of living organisms. Group I and Group II introns also make extensive internal interactions that allow them to fold into a specific, complex three-dimensional architecture. These complex architectures allow some group I and group II introns to be self-splicing, that is, the intron-containing RNA molecule can rearrange its own covalent structure so as to precisely remove the intron and link the exons together in the correct order. In some cases, particular intron-binding proteins are involved in splicing, acting in such a way that they assist the intron in folding into the three-dimensional structure that is necessary for self-splicing activity. Group I and group II introns are distinguished by different sets of internal conserved sequences and folded structures, and by the fact that splicing of RNA molecules containing group II introns generates branched introns, while group I introns use a non-encoded guanosine nucleotide (typically GTP) to initiate splicing, adding it on to the 5'-end of the excised intron.

2. Spliceosomal introns

Nuclear pre-mRNA introns are spliceosomal introns which characterized by specific intron sequences located at the boundaries between introns and exons. These sequences are recognized by spliceosomal RNA molecules when the splicing reactions are initiated. In addition, they contain a branch point, a particular nucleotide sequence near the 3' end of the intron that becomes covalently linked to the 5' end of the intron during the splicing process, generating a branched (lariat) intron. Apart from these three short conserved elements, nuclear pre-mRNA intron sequences are highly variable. Nuclear pre-mRNA introns are often much longer than their surrounding exons.

3. tRNA introns

Transfer RNA introns that depend upon proteins for removal occur at a specific location within the anticodon loop of unspliced tRNA precursors, and are removed by a tRNA splicing endonuclease. The exons are then linked together by a second protein, the tRNA splicing ligase.

Acquisition of introns in eukaryotic genes

Group II introns, are currently present in bacterial, mitochondrial, and chloroplast genomes, but absent from eukaryotic nuclear genomes. The surprising organization of eukaryotic genes in introns and exons evolved through the invasion of mobile genetic elements of group II introns. Group II introns act as RNA-based enzymes (ribozymes) that can self- splice and, therefore, eliminate themselves from the primary transcripts of their host genes. This, together with their ability to reverse splice and insert into new DNA sites, has facilitated their spread in genomes and thus played a critical role in the origin and evolution of eukaryotes. There are vast evidence argues for the common origin of group II and eukaryotic pre-mRNA introns.

1. Group II introns and eukaryotic pre-mRNA both are removed by same chemical mechanism comprising two consecutive SN2- type trans-esterification reactions involving a lariat intermediate harboring a 2’–5’ phosphodiester bond.
2. Striking similarities exist between the 3D architecture of the RNA at the catalytic core of group II introns and at the recently reported catalytic site of a yeast spliceosome.
3. A key common feature involves two Mg²⁺, coordinated by equivalent RNA phosphate moieties.Mg²⁺ activate the attacking group and stabilize the leaving group in each of the two phosphoryl-transfer steps of the splicing reaction a mechanism parallel to phosphoryl-transfer reactions of various protein enzymes.
   
   Molecular design of Spliceosome complex: The spliceosome can be envisioned as a highly sophisticated ribonucleoprotein chaperone that forces key sequences of the pre-mRNA into arrangements that enable the activation of an RNA catalytic center and consequent intron removal. This architecture comprise of various pieces which are subjected to timed compositional and conformational remodeling thus imparting structural flexibility to splicesomal complex.
   
   RNA Catalytic Core Embraced by a Protein Scaffold: After the first catalytic step,key intra-and intermolecular base pairing interactions involve three spliceosomal RNAs (U2,U5,and U6snRNAs) and part of the pre-mRNA. The pre- mRNA is held close to the spliceosome by base pairing interactions between the 5’end of the intron and U6 snRNA while the nucleotides flanking the branch point adenosine are base paired to U2 snRNA. These pre-mRNA snRNA contacts together with base pairing interactions between U2 and U6, provide the scaffold that holds the pre-mRNA onto the spliceosome and brings together the reacting groups. Base pairing interactions between U2 and U6 configure the active site. A particular key set of contacts involves a triple helix between a U6AGC triad, complementary nucleotides in U2 and nucleotides from distant regions of U6. As a result of this configuration, four phosphate residues are positioned such that they coordinate the two Mg²⁺ ions that facilitate phosphoryl- transfer reactions. The structure of this catalytic complex (known as complex C) is in fact an intermediate generated after the first step of the splicing reaction, which is to be remodeled to allow the second reaction step. Remodeling brings the 3’-OH at the end of the 5’ exon and the phosphate at the 3’end of the intron within the catalytic center. A key distinctive feature of the spliceosome is the fundamental role of proteins in configuring the ribozyme. In the ribosome, the other grand ribonucleoprotein ribozyme of the cell, RNA is responsible for the general organization of the complex and proteins occupy peripheral positions. As a result ribosome is RNA directed ribozyme. On the other hand spliceosomal proteins, in particular pre-mRNA-processing-splicing factor8 the most conserved protein in the spliceosome, are instrumental in organizing the RNA-based catalytic site and in regulating the conformational changes needed to activate catalysis. This suggests that Prp8 has evolved from these RNA chaperones and along with other protein partners orchestrate spliceosome assembly. The catalytic site docks on a cavity of Prp8, which stabilizes the structure. As a result electrostatic interactions form between a cluster of positively charged amino acids on the cavity surface and the internal stem loop of U6 which stabilize an RNA conformation relevant for catalysis. Further contact of PrP8 and other protein with loops of U5 snRNA confer it in rigid conformation which is necessary for providing a framework for holding the intermediates before the second step.
   
   An Open Catalytic Core: Prp8 and other proteins also contribute to another remarkable feature of the catalytic site. By interacting with other regions of the U2 and U6 snRNAs, these proteins ensure that these regions are kept away from the catalytic core. This open arrangement is likely to serve two distinct purposes. First, the open and peripheral location of the catalytic core makes it possible to accommodate the large variety of lengths which is characterstics of eukaryotic mRNA. Secondly an open catalytic site is also relevant for the coupling between splicing and other gene expression steps like transcription.
   
   Reconstructing the Broken Catalytic Heart: The catalytically active conformation of spliceosome is short lived and is reconstructed from individual pieces for each splicing event. This reconstruction is mainly for two needs of splicing machinery. First intron boundaries are often located far from each other in the pre-mRNA and therefore the two substrates of both trans-esterification reactions need to be brought near each other in space and time. A second consideration is that the active site is generated only transiently, after the specific reacting groups have been identified and brought together. Several protein interaction with snRNA is involved in reconstruction of catalytic site. The re-arrangement of base pairing interactions promoted by RNA helicases of the DExD/H box family which have key involvement in construction of catalytic site.
   
   Identifying the Correct Phosphates for Splicing Splice site sequences in multicellular organisms are highly degenerate. Spliceosomes are appreciated by the more alarming task of identifying multiple introns in each gene, commonly longer than flanking exon sequence. Further it is proposed that thousands of different sequences can function as 5’ splice sites or as branch points with only a few nucleotides being invariant. Therefore a proper mechanism should be present for correct identification of splicing sites. 5’splice sites are initially recognized by U1snRNP before base pairing with U6 snRNA. Base pairing between the 5’ end of U1 snRNA and the first six nucleotides of the intron, and up to three nucleotides of the exon, which is responsible for the specific sequence recognition. The possibility of using different base pairing registers and of accommodating nucleotides bulged out from the base paired region could allow the recognition of a variety of sequences. Stabilization of such mismatches is promoted by hydrogen bonds and electrostatic contacts between a protein components of U1and the RNA back bone at the 5’ splice-site region. Similar to 5’ splice -site recognition, 3’ splice-site recognition also depends critically on base pairing interactions between pre-mRNA and snRNAs. In this case, U2snRNA interacts with sequences flanking the branch point adenosine which is bulged out as a necessary signal for catalysis. Further splicing mechanism of nonconsensus intron sequences is under question and yet to be explored in future.

References:

1. The Spliceosome: The Ultimate RNA Chaperone and Sculptor: Trends in biochemical sciences- page 33-45. By P. Papasaikas, & J. Valcarcel. (2016)
2. Structural basis of pre-mRNA splicing: Science- Page 1191- 1198. By J. Hang, R. Wan, C. Yan & Y. Shi. (2015)


About Author / Additional Info:
Ph.D. Scholar, Biochemistry,
Indian Agricultural Research
Institute, New Delhi