Principles and correction of 5'-splice site selection

Abstract

In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon splicing, and further regulation of the retained exons leads to alternatively spliced mRNA. The splicing reaction requires the stepwise assembly of the spliceosome, a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs). This review focuses on the early stage of spliceosome assembly, when U1 snRNP defines each intron 5'-splice site (5'ss) in the pre-mRNA. We first introduce the splicing reaction and the impact of alternative splicing on gene expression regulation. Thereafter, we extensively discuss splicing descriptors that influence the 5'ss selection by U1 snRNP, such as sequence determinants, and interactions mediated by U1-specific proteins or U1 small nuclear RNA (U1 snRNA). We also include examples of diseases that affect the 5'ss selection by U1 snRNP, and discuss recent therapeutic advances that manipulate U1 snRNP 5'ss selectivity with antisense oligonucleotides and small-molecule splicing switches.

Keywords: 5’-splice site; RNA splicing; U1 snRNP; antisense oligonucleotides; splicing modifiers.

Figure 1.

Overview of the splicing reaction. The pre-mRNA must contain at least two exons (boxes) interleaved by one intron (black line) to be relevant for splicing. A single intron is defined by a pair of 5’- and 3’-splice sites (5ʹss, 3ʹss), as well as the branch point (BP) adenosine. Five small nuclear ribonucleoproteins (snRNPs) are the core components of the spliceosome machinery (U1/2/4/5/6) which requires auxiliary factors such as SF1 and U2AF. For each step in the diagram, the name of the corresponding spliceosomal complex is given.

Figure 2.

Alternative splicing events. Alternative splicing uses the intron 5’- and 3’-splice sites (3ʹss, 5ʹss), but with variations arising from different mechanisms: alternative first exon (AFE), alternative last exon (ALE), cassette exon, mutually exclusive exons, alternative 5ʹss and/or 3ʹss selection and intron retention.

Figure 3.

U1 snRNP structure and 5’-splice site (5ʹss) recognition. (a) Summary of U1 snRNP topology. U1 snRNP (light grey) is composed of the U1 snRNA (line), seven Sm proteins (dark grey) and three U1 snRNP specific proteins. (b) U1 snRNA sequence and topology. In U1 snRNA (164 nt), the 5’-end (red) binds to the 5ʹss, the downstream region contains SL1-3, followed by the Sm-site (grey), and SL4 in 3’-end. (c) Structural model of the complete U1 snRNP based on the crystal structure of human spliceosomal U1 snRNP [62]. U1 snRNA (light grey) and its 5’-end (red), the Sm-ring (grey) and U1-specific proteins U1-70 K (yellow), U1-A (pink) and U1-C (purple) are highlighted. (d) Base-pairing registers. U1 snRNA (red) and the 5ʹss (black) can adapt canonical and alternative registers.

Figure 4.

U1 snRNP-specific proteins interact with splicing factors to modulate 5’-splice site (5ʹss) recognition and spliceosome assembly. (a) Recruitment of U1 snRNP at the 5ʹss by SRSF1 upon interaction with U1-70 K [82]. The phosphorylation of ESE-bound SRSF1 in the RS domain makes its RRMs available for interaction with U1-70 K RRM, which contributes to recruiting U1 snRNP to the 5ʹss. (b) Sam68 interaction with U1 snRNP is mediated by U1-A to affect the definition of the alternative last exon [87,89]. The interaction between U1-A RRM1 and Sam68 YY-domain stabilizes the binding of U1 snRNP to the pre-mRNA, which in turn represses the polyadenylation signal and leads to inclusion of the terminal exon. In contrast, the absence of Sam68 does not allow U1 snRNP binding, which results in the inclusion of alternative last exon. (c) Recruitment of U1 snRNP at the 5ʹss by TIA-1 upon interaction with U1-C [94,95]. The binding of TIA-1 RRM1-2 to U-rich sequences downstream of the 5ʹss facilitates the recruitment of U1 snRNP through interactions between TIA-1 Q-rich domain and the U1-C protein.

Figure 5.

U1 snRNA SL3 and SL4 are targeted by splicing factors to modulate 5’-splice site (5ʹss) recognition and spliceosome assembly. (a) Protein cross-links to U1 snRNA in vivo [98,99]. The heat map shows the distribution of cross-links to U1 snRNA for 147 RNA-binding proteins. (b) Solution structure of FUS-RRM (blue) in complex with U1 snRNA SL3 (grey) (pdb code: 6SNJ) [101]. The structure corresponds to the lowest energy model from the NMR ensemble. (c) Model for exon independent recruitment of SRSF1 by U1 snRNP [98]. (d) Model for UAP56 mediated splicing enhancement [100]. (e) Crystal structure of SF3A1-UBL (cyan) in complex with U1 snRNA SL4 (grey) (pdb code: 7P0V) [108]. (f) Model for PTB mediated splicing repression [110,111].

Figure 6.

Antisense oligonucleotide (ASO) can modulate 5’-splice site (5ʹss) strength by masking nearby cis-regulatory sequences. (a) Exon skipping. ASO can mask enhancer sequences, promoting exon skipping. (b) Exon inclusion. ASO can mask silencer sequences, promoting exon inclusion. (c) RNA modification. Phosphorothioate (PS) backbone, 2’-O-methoxyethyl (2’-MOE), and phosphorodiamidate morpholino oligomer (PMO).

Figure 7.

Small-molecule splicing modifiers can strengthen 5’-splice site (5ʹss) interaction with U1 snRNP through the mechanism of bulge-repair. (a) Chemical structure of Risdiplam, Branaplam, and analogues for the treatment of spinal muscular atrophy (SMA) and Huntington disease. (b) Solution structures of U1 snRNA 5’-end (grey) in complex with the 5ʹss (blue) of SMN exon 7, in absence (left, pdb code: 6HMI) and in presence (right, pdb code: 6HMO) of SMNC5 (yellow) [101]. Within the 5ʹss, the A-1 (red) bulging out in absence of small molecule (left) turns inward when SMN-C5 is present (right). (c) The bulge-repair concept. A weak 5ʹss may be strengthened by small-molecule splicing modifiers acting as a glue between U1 snRNP and the 5ʹss.