RNA Splicing

Lecture Objectives

Describe the components and mechanisms of splicing.
Explain the importance of the 5’ cap and how it is added.
Describe the major and minor splice sites, how they are read, and their role in splicing.
Explain the formation of the lariat.
Differentiate between different types of RNAs discussed in lecture and explain the role of snRNAs and snurps.
Outline the steps of the splicing and spliceosome pathway and alternative pathways.
Explain how some introns are self-splicing and describe cis and trans splicing mechanisms.
Explain the importance and function of the EJC.
Describe alternative splicing, its importance, and its mechanisms.
Describe splicing enhancers and silencers and their roles in alternative splicing.
Describe the role of splicing in tRNA and rRNA formation.

19.1 Introduction

pre-mRNA: The nuclear transcript that is processed by modification and splicing to give an mRNA.
RNA splicing: The process of excising introns from RNA and connecting the exons into a continuous mRNA.
Spliceosome: Splicing complex composed of protein and RNAs (including mRNA). Holds exons together for proper organization.
RNA modification occurs in the nucleus involving additions to the 5’ and 3’ ends, and splicing to remove introns.
heterogeneous nuclear RNA (hnRNA): RNA that comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability.
hnRNP: The ribonucleoprotein form of hnRNA (heterogeneous nuclear RNA), in which the hnRNA is complexed with proteins.
Pre-mRNAs are not exported until processing is complete; thus, they are found only in the nucleus.

19.2 The 5′ End of Eukaryotic mRNA Is Capped

A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–5′ link.
The capping process takes place during transcription and may be important for release from pausing of transcription.
The cap blocks the 5’ end of mRNA and is methylated at several positions.
The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated.
The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation.

19.3 Nuclear Splice Sites Are Short Sequences

Splice sites are the sequences immediately surrounding the exon–intron boundaries. They are named for their positions relative to the intron.
The 5′ splice site at the 5′ (“left”) end of the intron includes the consensus sequence GU.
The 3′ splice site at the 3′ (“right”) end of the intron includes the consensus sequence AG.
The GU-AG rule (originally called the GT-AG rule in terms of DNA sequence) describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs.
The ends of nuclear introns are defined by the GU-AG rule.
Minor introns exist relative to the major introns that follow the GU-AG rule.
Minor introns follow a general AU-AC rule with a different set of consensus sequences at the exon–intron boundaries.

19.4 Splice Sites Are Read in Pairs

Splicing depends only on recognition of pairs of splice sites.
All 5′ splice sites are functionally equivalent, as are all 3′ splice sites.
Additional conserved sequences at both 5′ and 3′ splice sites define functional splice sites among numerous other potential sites in the pre-mRNA.
Splicing junctions are recognized only in the correct pairwise combinations.

19.5 Pre-mRNA Splicing Proceeds Through a Lariat

Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site.
The branch sequence is conserved in yeast but less well conserved in multicellular eukaryotes.
A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at an A at the branch site in the intron.
The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together.
Splicing occurs in two stages: first, the 5’ exon is cleaved off, and then it is joined to the 3’ exon.
Nuclear splicing occurs through two transesterification reactions, where an -OH group attacks a phosphodiester bond.

19.6 snRNAs Are Required for Splicing

small cytoplasmic RNAs (scRNA; scyrps): RNAs that are present in the cytoplasm (and sometimes are also found in the nucleus).
small nuclear RNA (snRNA; snurps): One of many small RNA species confined to the nucleus; several of them are involved in splicing or other RNA processing reactions.
small nucleolar RNA (snoRNA): A small nuclear RNA that is localized in the nucleolus.
The five snRNPs involved in splicing are U1, U2, U5, U4, and U6.
Together with some additional proteins, the snRNPs form the spliceosome.
The spliceosome is ~12 MDa; five snRNPs account for almost half of the mass.
All the snRNPs except U6 contain a conserved sequence that binds the Sm proteins that are recognized by antibodies (anti-SM) generated in autoimmune disease.
splicing factor: A protein component of the spliceosome that is not part of one of the snRNPs.

19.7 Commitment of Pre-mRNA to the Splicing Pathway

U1 snRNP initiates splicing by binding to the 5′ splice site by means of an RNA–RNA pairing reaction.
The commitment complex (or E complex) contains U1 snRNP bound at the 5′ splice site and the protein U2AF bound to a pyrimidine tract between the branch site and the 3′ splice site.
U1 snRNA has a base-paired structure that creates several domains; the 5’ end remains single-stranded and can base pair with the 5’ splice site.
In cells of multicellular eukaryotes, SR proteins play an essential role in initiating the formation of the commitment complex.
SR proteins have RNA binding motifs that are sequence-specific.
Pairing splice sites can be accomplished by intron definition or exon definition.

19.8 The Spliceosome Assembly Pathway

The commitment complex progresses to prespliceosome (the A complex) in the presence of ATP.
Recruitment of U5 and U4/U6 snRNPs converts the A complex to the mature spliceosome (the B1 complex).
The B1 complex is next converted to the B2 complex, in which U1 snRNP is released to allow U6 snRNA to interact with the 5′ splice site.
When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytically active site.
Both transesterification reactions take place in the activated spliceosome (the C complex).
The splicing reaction is reversible at all steps.
The splicing reaction proceeds through discrete stages.

19.9 An Alternative Spliceosome Uses Different snRNPs to Process the Minor Class of Introns

An alternative splicing pathway uses another set of snRNPs that comprise the U12 spliceosome.
The target introns are defined by longer consensus sequences at the splice junctions rather than strictly according to the GU-AG or AU-AC rules.
Major and minor spliceosomes share critical protein factors, including SR proteins.

19.10 Pre-mRNA Splicing Likely Shares the Mechanism with Group II Autocatalytic Introns

Group II introns excise themselves from RNA by an autocatalytic splicing event (autosplicing or self-splicing).
The splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns.
A group II intron folds into a secondary structure that generates a catalytic site resembling the structure of U6-U2 nuclear intron.

19.11 Splicing Is Temporally and Functionally Coupled with Multiple Steps in Gene Expression

Splicing can occur during or after transcription.
The transcription and splicing machineries are physically and functionally integrated.
Splicing is connected to mRNA export and stability control.
exon junction complex (EJC): A protein complex that assembles at exon–exon junctions during splicing and assists in RNA transport, localization, and degradation.
The EJC (exon junction complex) is deposited near the splice junction as a consequence of the splicing reaction.
Splicing in the nucleus can influence mRNA translation in the cytoplasm.
nonsense-mediated mRNA decay (NMD): A pathway that degrades an mRNA that has a nonsense mutation prior to the last exon.
The EJC complex couples splicing with NMD.

19.12 Alternative Splicing Is a Rule, Rather Than an Exception, in Multicellular Eukaryotes

Specific exons or exonic sequences may be excluded or included in the mRNA products by using alternative splicing sites.
Alternative splicing contributes to structural and functional diversity of gene products.
Different modes of alternative splicing exist.

19.13 Splicing Can Be Regulated by Exonic and Intronic Splicing Enhancers and Silencers

Alternative splicing is often associated with weak splice sites.
Sequences surrounding alternative exons are often more evolutionarily conserved than sequences flanking constitutive exons.
Specific exonic and intronic sequences can enhance or suppress splice-site selection.
Exonic and intronic sequences can modulate the splice site selection by functioning as splicing enhancers or silencers.
The effect of splicing enhancers and silencers is mediated by sequence-specific RNA binding proteins, many of which may be developmentally regulated and/or expressed in a tissue-specific manner.
The rate of transcription can directly affect the outcome of alternative splicing.
The Nova and Fox families of RNA binding proteins can promote or suppress splice site selection in a context-dependent fashion.

19.14 trans-Splicing Reactions Use Small RNAs

Splicing reactions usually occur only in cis between splice sites on the same molecule of RNA.
trans-splicing occurs in trypanosomes and worms where a short sequence (SL RNA) is spliced to the 5′ ends of many precursor mRNAs.
Splicing usually occurs only in cis between exons carried on the same physical RNA molecule.

19.18 tRNA Splicing Involves Cutting and Rejoining in Separate Reactions

RNA polymerase III terminates transcription in a poly(U)4 sequence embedded in a GC-rich sequence.
tRNA splicing occurs by successive cleavage and ligation reactions.
The intron in yeast tRNAPhe base pairs with the anticodon to change the structure of the anticodon arm.
Splicing of tRNA requires separate nuclease and ligase activities.

19.19 The Unfolded Protein Response Is Related to tRNA Splicing

Ire1 is an inner nuclear membrane protein with its N-terminal domain in the ER lumen and its C-terminal domain in the nucleus; the C-terminal domain exhibits both kinase and endonuclease activities.
Binding of an unfolded protein to the N-terminal domain activates the C-terminal endonuclease by autophosphorylation.
The activated endonuclease cleaves HAC1 (Xbp1 in vertebrates) mRNA to release an intron and generate exons that are ligated by a tRNA ligase.
Only spliced HAC1 mRNA can be translated to a transcription factor that activates genes encoding chaperones that help to fold unfolded proteins.
Activated Ire1 induces apoptosis when the cell is overstressed by unfolded proteins.

19.20 Production of rRNA Requires Cleavage Events and Involves Small RNAs

RNA polymerase I terminates transcription at an 18-base terminator sequence.
The large and small rRNAs are released by cleavage from a common precursor rRNA; the 5S rRNA is separately transcribed.
Mature eukaryotic rRNAs are generated by cleavage and trimming events from a primary transcript.