Lariat Structure in Eukaryotic Introns: A Closer Look

Introns in eukaryotic genes are removed through a precise and highly regulated process known as splicing. Many introns form an intermediate structure called a lariat, which plays a crucial role in ensuring accurate RNA processing before degradation or further utilization. Understanding lariat formation and function provides insight into gene regulation, alternative splicing, and potential implications for genetic disorders.

Despite their transient nature, lariat structures have been studied using various molecular and structural techniques to uncover their properties. Researchers continue to explore how sequence elements, biochemical pathways, and species-specific variations influence lariat formation and stability.

Key Molecular Features

The lariat structure formed during eukaryotic pre-mRNA splicing is defined by a unique 2′-5′ phosphodiester bond linking the branch point adenosine to the 5′ splice site. This covalent linkage creates a looped configuration, distinguishing lariats from linear RNA intermediates. The stability and formation of this structure depend on precise molecular interactions, including conserved sequence motifs and spliceosome activity.

The branch point sequence, typically 18–40 nucleotides upstream of the 3′ splice site, plays a central role by providing the nucleophilic adenosine that initiates the first transesterification reaction. Mutations in this sequence can significantly alter splicing efficiency and lariat stability, sometimes leading to exon skipping or cryptic splice site activation (Taggart et al., 2017, Genome Research).

The 5′ splice site contributes by providing the donor exon-intron junction necessary for the first step of splicing. This region is initially recognized by U1 small nuclear RNA (snRNA) and later displaced by U6 snRNA. The strength of the 5′ splice site, determined by its complementarity to U1 and U6, influences lariat formation efficiency. Weak splice sites often require additional splicing enhancers or auxiliary proteins.

The polypyrimidine tract and the 3′ splice site, though primarily involved in exon definition, indirectly affect lariat stability by modulating spliceosome dynamics. Additionally, certain introns contain RNA folding elements, such as stem-loops or G-quadruplexes, that can enhance or hinder splicing efficiency. SHAPE probing has shown that some lariats adopt compact conformations that protect them from rapid degradation, while others remain more exposed to exonucleases (Eryilmaz et al., 2020, RNA).

Sequence Elements Influencing Lariat Structure

Lariat formation and stability are governed by specific sequence elements that dictate spliceosome assembly and catalytic efficiency. The branch point sequence (BPS) serves as the defining nucleotide motif anchoring lariat formation. The conserved adenosine residue within the BPS acts as the nucleophile in the first transesterification reaction. Variations in these sequences can disrupt spliceosome recognition or alter splicing kinetics, reducing fidelity and leading to exon skipping or cryptic splice site activation (Mercer et al., 2015, Nature Communications).

The 5′ splice site contributes to lariat topology by providing the donor junction for the first step of splicing. This region contains a conserved GU dinucleotide at the intron boundary, recognized by U1 and later U6 snRNAs. The strength of the 5′ splice site influences splicing efficiency, with weaker sites often requiring regulatory elements. Mutations in this region can lead to aberrant splicing outcomes, including retained introns or alternative exon usage (Wang et al., 2019, Molecular Cell).

The polypyrimidine tract (PPT) and the 3′ splice site shape lariat stability by modulating spliceosome interactions. The PPT, enriched in uridine residues, serves as a binding platform for splicing factors such as U2AF65. Variability in PPT length and composition affects lariat formation, with shorter or disrupted tracts leading to inefficient splicing (Hollander et al., 2016, RNA Biology).

RNA secondary structures within introns influence lariat loops. Some introns contain stem-loop structures or G-quadruplex motifs that affect splice site accessibility and lariat formation kinetics. In vitro splicing assays have shown that intronic stem-loops near the branch point can enhance lariat stability by reducing exonucleolytic degradation, whereas misfolded RNA structures can impede spliceosome assembly (Khodor et al., 2011, Genes & Development).

Biochemical Pathways Forming Lariats

Lariat formation follows a sequence of biochemical reactions driven by the spliceosome. This ribonucleoprotein complex undergoes conformational changes to facilitate the two-step transesterification process essential for intron removal.

Splicing begins with the recognition of the 5′ splice site and branch point sequence by U1 and U2 snRNPs, guiding the recruitment of the U4/U6.U5 tri-snRNP to form the precatalytic spliceosome (complex B). ATP-dependent RNA helicases, such as Prp5 and Sub2, remodel RNA-protein interactions to ensure proper splice site alignment before catalysis (Wahl et al., 2009, Cell).

As the spliceosome transitions into its active state (complex B), U1 and U4 snRNPs dissociate, allowing U6 snRNA to pair with the 5′ splice site. This rearrangement positions the catalytic core, where U2 snRNA base-pairs with the branch point adenosine, priming it for nucleophilic attack. The first transesterification reaction forms the 2′-5′ lariat linkage and releases the upstream exon, facilitated by Prp2, a DEAH-box ATPase (Fica et al., 2013, Nature).

The second transesterification step joins the exons while releasing the intron in its lariat form. The post-splicing complex, containing the excised lariat, remains associated with spliceosomal proteins until disassembly is triggered by Prp22. The lariat is subsequently debranched by the Dbr1 enzyme, converting it into a linear RNA for degradation. Defects in Dbr1 activity have been linked to neurodegenerative disorders due to lariat-derived RNA accumulation (Han et al., 2020, Neuron).

Structural Approaches

Understanding lariat structure has been challenging due to its transient nature and susceptibility to degradation. Advances in structural biology have provided insights into its topology and interactions with spliceosomal components.

X-Ray Crystallography

X-ray crystallography has resolved high-resolution structures of spliceosomal components, though capturing intact lariats remains difficult due to their dynamic nature. This technique relies on crystallizing RNA-protein complexes and analyzing diffraction patterns. While full lariat structures have not been crystallized, key spliceosomal proteins, such as Prp8, have been studied. A 2015 Nature study detailed Prp8’s RNase H-like domain, revealing how it stabilizes the branch point adenosine during the first transesterification reaction.

Cryo-EM

Cryo-electron microscopy (cryo-EM) has revolutionized spliceosomal studies by visualizing ribonucleoprotein complexes at near-atomic resolution. Unlike X-ray crystallography, cryo-EM does not require crystallization, making it ideal for studying transient RNA structures such as lariats. A 2021 Science study presented a 3.3 Å resolution structure of the post-catalytic spliceosome, showing how the lariat intron remains stabilized by U2 and U5 snRNPs before disassembly. These findings provide unprecedented insights into the conformational changes during lariat formation and release.

Other Techniques

Other biophysical and biochemical methods have been used to study lariats. SHAPE probing has revealed how lariat loops adopt compact or extended conformations depending on sequence context. Crosslinking and immunoprecipitation (CLIP) techniques have identified protein-RNA interactions stabilizing lariats. Single-molecule Förster Resonance Energy Transfer (smFRET) has provided real-time insights into dynamic lariat rearrangements, highlighting the role of ATP-dependent helicases.

Variation in Different Eukaryotes

Lariat formation varies across eukaryotic species due to differences in spliceosome composition, intron architecture, and regulatory mechanisms. Yeast introns are relatively rare and short, with highly conserved branch point sequences that facilitate efficient spliceosome assembly. The Saccharomyces cerevisiae spliceosome is streamlined compared to metazoan counterparts, relying on fewer auxiliary factors (Schwer et al., 2016, RNA).

In contrast, multicellular eukaryotes exhibit greater complexity due to alternative splicing and longer introns with diverse sequence motifs. Human introns can span thousands of nucleotides, often containing multiple branch points that contribute to alternative splicing regulation. The presence of weak splice sites necessitates splicing enhancers and silencers. Some species, such as Drosophila melanogaster, utilize non-canonical splicing mechanisms generating atypical lariat structures. In plants, intron retention is a common regulatory strategy, with some lariats persisting longer before debranching (Marquez et al., 2012, Genome Biology). These variations highlight the evolutionary adaptability of splicing mechanisms across species.