RNA Splicing Steps: Detailed Analysis And Facts

Splicing is the process of converting hetero-nuclear RNA to messenger RNA in the eukaryotic central dogma.

RNA splicing is a mechanism in which genetic information is changed while in RNA-form during eukaryotic gene expression. The process is called post-transcriptional processing i.e it is a part of RNA transcription from the gene.

An explanation of the transmission of genetic information inside a biological system is the core tenet of molecular biology. Although this is not its original meaning, it is sometimes phrased as “DNA produces RNA, and RNA makes protein.” This is the core mechanism o the Central Dogma.

Simplistically it means conversion of DNA(gene) to RNA to protein via transcription and translation and occurs in both prokaryotes and eukaryotes. The RNA splicing steps are discussed below.

RNA splicing definition:

In terms of molecular biology, RNA splicing is one of the processes involved in converting an mRNA precursor to a mature mRNA. This is done by removing the introns or the non-coding genes in the pre-mRNA or hnRNA and joining only the coding genes or exons to form the mRNA chain.

For genes encoded in the nucleus as in eukaryotes, splicing occurs immediately after transcription and is called a post-transcriptional process.

RNA splicing mechanism:

The process of splicing occurs in several steps. The RNA splicing steps are:

  • Step1: Formation and activation of different spliceosome complexes
  • Step2: Finding the starting and ending points of the introns and removing them
  • Step3: Joining the exons together.
An illustration showing RNA splicing steps
Image: Wikipedia

Formation of the spliceosome and the spliceosome location the introns and cutting them off occur simultaneously in the same step, followed by the joining of the exons.


Splicing in hnRNA is initiated by the spliceosome, a large RNA-protein complex made up of five small nuclear ribonucleoproteins (snRNPs). The spliceosome is assembled and activated during the transcription of the hnRNA. SnRNPs’ RNA components interact with the intron and have a role in catalysis. There are two kinds of spliceosomes (major and minor) that contain various snRNPs.

In this process, 2 types of spliceosomes are produced- the major spliceosome and the minor spliceosome. These two types of spliceosomes have distinctly different functions.


Major spliceosome:

The Major spliceosome splices introns with G(Guanine)U(Uracil) sequence at the 5′ splice site and A(Adenine)G(Guanine) sequence at the 3′ splice site. It is active in the nucleus and is made up of the 6 snRNPs-U1, U2, U4, U5, and U6. Spliceosome formation also requires several proteins, including U2 small nuclear RNA auxiliary factor 1 (U2AF35), U2AF2 (U2AF65), and SF1 (Splicing Factor 1). During the process of RNA splicing, several complexes with diverse functions are produced by the spliceosome including:

Intron miguelferig 1
Image showing the intron sequence between the exons
Image: Wikipedia

Complex E:

  • U1 snRNP goes and binds to the GU sequence in an intron’s 5′-splice site
  • Splicing factor1(SF1) binds to the same intron’s branchpoint sequence;
  • U2AF1 binds to the splice site present on the 3’ end of the intron;
  •  U2AF2 goes to bind itself to the polypyrimidine tract;

Complex A (pre-spliceosome complex)

  • The U2 snRNP attaches to the branch point sequence and displaces Splicing Factor 1, causing ATP to be hydrolyzed.

Complex B

  • Three snRNPs-U5, U4 and U6 bind together to form a trimeric complex, where the U5 snRNP binds to exons at the 5′ site while U6 binds to U2.

Complex B*:

  • The U1 snRNP complex is released. After the positions of the U5 shift from the exon to the intron, the U6 goes and binds to the 5′ splice site that was previously occupied by the U5.

Complex C (catalytic spliceosome):

  • The U4 snRNP is released At the same time the U6/U2 catalyses transesterification(exchanging the organic group R” group of an ester molecule with the organic group R’ group of an alcohol molecule).
  • The 5′ end of the intron goes around to ligate to the Adenine on itself, forming a lariat; the U5 binds exon at the 3′ splice site, and the 5′ site is cleaved, forming the lariat; and the U5 binds exon at the 3′ splice site, causing the lariat to form.

Canonical splicing, also known as the lariat route, is the most common kind of splicing that occurs in nature. It accounts for more than 99% of all splicing that occurs in all RNA varieties.

When the sequences flanking on the sides of the intron do not obey the GU-AG (GuanineUracil- GuanineAdenine) rule, it is referred to as noncanonical splicing.

Minor spliceosome
Image comparing splicing between major and Minor spliceosome
Image: Wikipedia

Minor spliceosome:

The function of the minor spliceosome is quite similar to that of the major spliceosome, but it splices out uncommon introns with distinct splice site sequences. While the U5 snRNP is similar in both the minor and major spliceosomes, the minor spliceosome possesses distinct but functionally analogous snRNPs for U1, U2, U4, and U6, known as U11, U12, U4atac, and U6atac respectively.


This involves Complex C* which is a post-spliceosomal complex. The last step of splicing constitutes the cleaving of the  3′ site and litigation of the exons utilising ATP hydrolysis, while U2/U5/U6 remain attached to the lariat. The spliced RNA, the lariat, and the snRNPs are all released and destroyed before being recycled.

And hence we get mRNA free of introns and only constituting only of coding introns capped and tailed at the 5’ and 3’ ends respectively.

What happens in RNA splicing?

The first RNA transcribed from a gene’s DNA template must be processed before it becomes a mature messenger RNA (mRNA) that can control protein synthesis in most eukaryotic genes (and some prokaryotic genes).

Most eukaryotic genes (and some prokaryotic genes as well) require processing before the pre-messenger RNA is converted into a mature messenger RNA (mRNA) that can actually be used for synthesizing protein.

1920px Spliceosome ball cycle new2
Illustration of the spliceosome cycle
Image: Wikipedia

The processing of hnRNA to mature mRNA requires 3 steps in total:

  • Addition of 7-methyl guanosine at the 5’ end
  • Trimming of the 3’ end and addition of a 200 “A” nucleotide tail by an enzyme called Poly A Polymerase.
  • Splicing of the introns.

So we see that splicing is only one of the steps of post-transcriptional processing of RNA.

How does RNA splicing work?

Splicing’s biochemical mechanism has been researched in a variety of situations and is now pretty well described.

Introns are eliminated from main transcripts by cleavage at splice sites, which are conserved sequences. Introns have these locations at the 5’- and 3”- ends. The RNA sequence most typically deleted starts with the dinucleotide GU at the 5’-end and finishes with AG at the 3’-end.

Even if a single nucleotide is altered, it can inhibit the entire splicing process; hence conserving the consensus nucleotide sequence is important. Another significant region occurs at the branch point, which may be found anywhere between 18 and 40 nucleotides upstream from an intron’s 3’-end. The adenine at the branch point is always present, while the rest of the sequence is rather weakly preserved.

Splicing is carried out in numerous phases, with tiny nuclear ribonucleoproteins acting as catalysts (snRNPs, commonly called “snurps“).

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