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Updated: Jan 18, 2026
Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
Published on: June 24, 2021
Molecular basis for the recognition of low-frequency polyadenylation signals by mPSF
Lin Huang1, Hsu-Feng Chu1, Liang Tong1
1Department of Biological Sciences, Columbia University, New York, NY 10027, United States.
Abstract:
The 3'-end cleavage and polyadenylation of pre-mRNAs is dependent on a key hexanucleotide motif known as the polyadenylation signal (PAS). The PAS hexamer is recognized by the mammalian polyadenylation specificity factor (mPSF). AAUAAA is the most frequent PAS hexamer and together with AUUAAA, the second most frequent hexamer, account for ∼75% of the poly(A) signals. The remaining hexamers are at low frequency (<3%), and the molecular basis for their recognition is still not known. Here, we have determined the binding affinities for most of the PAS hexamers, showing that the Kd values are generally inversely correlated with their frequency. We also observed good cleavage activity for two low-frequency hexamers, AAGAAA and AACAAA. We have determined the cryo-electron microscopy structures of human mPSF in complex with AAUAAU and AGUAAA, at 3.1 and 2.5 Å resolution, respectively. The overall binding modes of the two low-frequency hexamers are similar to that of AAUAAA, although the U3-A6 Hoogsteen base pair is disrupted in the AAUAAU hexamer. For AGUAAA, the G2 base undergoes a large conformational change, which allows it to maintain the hydrogen-bonding interaction with CPSF30 as observed with A2 and establish a new hydrogen bond to CPSF30.
Insights
The study reveals how low-frequency polyadenylation signals are recognized by mammalian polyadenylation specificity factor (mPSF). Structural analysis shows distinct binding modes for these signals, explaining their lower occurrence in gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Pre-mRNA 3'-end processing relies on the polyadenylation signal (PAS), a hexanucleotide motif.
- Mammalian polyadenylation specificity factor (mPSF) recognizes the PAS hexamer.
- While AAUAAA and AUUAAA dominate, the recognition of low-frequency PAS hexamers remains unclear.
Purpose of the Study:
- To investigate the molecular basis for the recognition of low-frequency polyadenylation signals by mPSF.
- To determine the binding affinities and structural basis for mPSF interaction with various PAS hexamers.
Main Methods:
- Determination of binding affinities (Kd values) for multiple PAS hexamers.
- Cryo-electron microscopy (cryo-EM) to resolve structures of human mPSF bound to AAUAAU and AGUAAA at high resolution (3.1 Å and 2.5 Å).
Main Results:
- Binding affinities generally inversely correlate with PAS hexamer frequency.
- Observed good cleavage activity for low-frequency hexamers AAGAAA and AACAAA.
- Cryo-EM structures revealed that low-frequency hexamers bind similarly to AAUAAA, with specific base modifications (e.g., disrupted Hoogsteen base pair in AAUAAU, conformational change in AGUAAA's G2 base) enabling interaction with CPSF30.
Conclusions:
- The study elucidates the molecular mechanisms underlying the recognition of diverse polyadenylation signals by mPSF.
- Structural insights explain how low-frequency PAS variants maintain functional interactions, contributing to the complexity of gene expression regulation.
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