mRNA SPLICING

M.Sc 4th semester

PG BOTANY DEPARTMENT

GAYA COLLEGE GAYA

DATE: 18th and 19th sep 2020

Dr Moni Kumari

Exons are coding sections of an RNA transcript

Or

An is the region of a that contains the code for producing a protein.

Most contain many , with each exon containing the information for a specific portion of a complete protein.

In many species, a gene's exons are separated by long regions of DNA that have no identified function. These long regions are , and must be removed prior to .

RNA splicing is a process in eukaryotic gene expression where that genetic information is altered while in RNA form.

In splicing, specific regions of the RNA transcript are cut out and the flanking sequences are pasted together.

The process of splicing fundamentally changes the information content of the RNA transcript, which directly impacts translation of that genetic information into protein.

Regulation of splicing therefore represents a critical step of gene expression

Sometimes a process called alternative splicing allows pre-mRNA messages to be spliced in several different configurations, allowing a single gene to encode multiple proteins.

Alternate splicing a strategy frequently used by higher eukaryotic cells to increase proteome diversity and/or enable additional post-transcriptional control of gene expression. This process can take place either co-transcriptionally or post-transcriptionally.

In prokaryotes, splicing is a rare event that occurs in non-coding RNAs, such as tRNAs (22)

Mainly, Splicing is only done in eukaryotes. Since they don't process mRNA by splicing, prokaryotes don't really have exons or introns.

Bacterial mRNAs do not have introns.

Splicing makes genes more "modular," allowing new combinations of exons to be created during evolution. Furthermore, new exons can be inserted into old introns, creating new proteins without disrupting the function of the old gene.

Gene splicing technology, therefore, allows researchers to insert new genes into the existing genetic material of an organisms genome so that entire traits, from disease resistance to vitamins, and can be copied from one organism and transferred another.

In eukaryotic cells, pre-mRNAs undergo three main processing steps:

 Capping at the 5' end.

 Addition of a polyA tail at the 3' end. and.

 Splicing to remove introns.

Once the mRNA has been capped, spliced and had a polyA tail added, it is sent from the nucleus into the cytoplasm for translation.

Several signals exist within introns that are critical for the splicing process including a 5′ splice site, a branch site, and a 3′ splice site

The splice sites are located at the 5′ and 3′ ends of introns and contain almost invariant sequence: GU at the 5′ splice site and AG at the 3′ splice site

Splicing is usually performed by an RNA-protein complex called the spliceosome, but some RNA molecules have their own catalytic activity and are capable of acting like enzymes to catalyze their own splicing.

A SPLICEOSOME is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells.

Spliceosomes are multimegadalton RNA–protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs)

In spliceosomes mainly 5 RNAs and many proteins are present collectively called small nuclear ribonucleoproteins or snRNPs (pronounced SNURPS).

They assemble on RNA polymerase II transcripts from which they excise RNA sequences called introns and splice together the flanking sequences called exons.

The snRNA component of the snRNP gives specificity to individual introns by "recognizing" the sequences of critical splicing signals at the 5' and 3' ends and branch site of introns.

Every human cell contains ~100,000 spliceosomes, which are responsible for removing over 200,000 different sequences

Human cells contain two types of spliceosome: the major spliceosome responsible for removing 99.5% of introns and the minor spliceosome, which removes the remaining 0.5%.

Many of these proteins (of spliceosome) have specific RNA recognition activities, while others are NTPases that function to drive the overall process forward and ensure its fidelity. Numerous other proteins bind stably to small nuclear RNAs (snRNAs) to form small nuclear RNPs (snRNPs, pronounced ‘snurps’). Major spliceosomes are assembled from U1, U2, U4, U6, and U5 snRNPs (which are named according to the U snRNA(s) they contain); minor spliceosomes are assembled from U11, U12, U4atac, U5, and U6atac snRNPs

Lariat Structures

Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve reactions that occur between RNA nucleotides. First, the 2'-OH of a exon-intron junction nucleotide within the intron binds to the first nucleotide of the intron at the 5' splice site, forming an intermediate known as a lariat. Second, the 3'-OH of the released 5' exon then binds to the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat.

Fig: Two step chemistry of mRNA splicing

5' cap Addition Capping is the first modification made to RNA polymerase II-transcribed RNA and takes place co-transcriptionally in the nucleus as soon as the first 25–30 nts are incorporated into the nascent transcript Question: How does the mRNA know it is time to leave the nucleus? Once the mRNA leaves the nucleus, how does it find a ?

Answer: A signal on the front, 5’-end of the mRNA helps with both jobs. This signal is the 5’ cap. The 5' cap is a modified guanine nucleotide ( a 7-methylguanosine) added to the 5’-end of the pre-mRNA soon after the start of . This 5’ cap is crucial for recognition and proper attachment of the mRNA to the ribosome, as well as protection from exonucleases, enzymes that degrade nucleic acids. Specifically the 5' cap adds stability from RNases. The process of 5' capping is vital to creating mature messenger RNA prior to translation.

The cap is also known as a 7-methylguanosine cap, abbreviated m7G.

The 5′ m7G cap is an evolutionarily conserved modification of eukaryotic mRNA.

Figure: 5' (7-methylguanosine) cap structure

Editing Most often this involves the editing or modification of one base to another, but in some organisms can involve the insertion or deletion of a base. Such editing events alter the coding properties of mRNA.

RNA editing can be generally defined as the co- or post transcriptional modification of the primary sequence of RNA from that encoded in the genome through nucleotide deletion, insertion, or base modification mechanisms. There are two pathways of RNA editing: the substitution/conversion pathway and the insertion/deletion pathway.

In certain instances, the nucleotide sequence of an mRNA will be changed to allow the mRNA to produce multiple proteins. This process is called editing. The classic example is editing of the apolipoprotein B (APOB) mRNA in humans. APOB is a protein that is responsible for carrying cholesterol to tissues. It is the primary lipoprotein of LDL (low density lipoprotein).

The APOB protein occurs in the plasma in two main forms, APOB48 and APOB100. APOB48 has 48% of the molecular weight has APOB100. The first is synthesized exclusively by the small intestine, the second by the liver. Both proteins are coded for by the same gene, which is transcribed into a single pre-mRNA. Editing changes a C to a U in the mRNA, changing a CAA codon (Glutamine) to a UAA codon, which is a premature (early) stop codon. Hence, upon translation this base change results in a smaller protein. As a result of the RNA editing, APOB48 and APOB100 share a common N-terminal sequence, but APOB48 lacks APOB100's C-terminal region. APOB48 contains only the first 2152 amino acids of the full-length 4536 amino acid APOB100.

Polyadenylation In eukaryotic cells, the transcription of the signals indicates the termination of the process. The mRNA transcript is then cut off of the RNA polymerase and freed from the DNA. The cleavage site is characterized by the presence of the sequence AAUAAA near the end of the transcribed message. Polyadenylation then occurs. Polyadenylation is the addition of a poly (A) tail to the 3’-end of the mRNA. The poly (A) tail may consist of as many as 80 to 250 adenosine residues. The poly (A) tail protects the mRNA from degradation by exonucleases. Poladenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation.

FUNCTION OF POLYADENYLATION: In nuclear polyadenylation, a poly (A) tail is added to an RNA at the end of transcription. On mRNAs, the poly (A) tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation.

Polyadenylation is a postranscriptional modification of RNA found in all cells and in organelles.