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Short poly(A) tails are protected from deadenylation by the LARP1–PABP complex

Nature Structural & Molecular Biology volume 30, pages 330–338 (2023)Cite this article

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Abstract

Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30–60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30–60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30–60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4–NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.

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Fig. 1: Steady-state and dynamic poly(A) length distributions.
Fig. 2: Effective ranges of LARPs on the poly(A) tail.
Fig. 3: Loss of LARP1 accelerates deadenylation in cells.
Fig. 4: LARP1 protects mRNAs with 30–60 nt of poly(A) tails globally.
Fig. 5: LARP1 directly blocks the deadenylation by CCR4–CAF1 in vitro.
Fig. 6: Model for decelerated deadenylation by LARP1 barricade on short poly(A) tails.

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Data availability

Hire-PAT, BPA, SEC, double-filter binding assay and mTAIL-seq Tailseeker-processed data have been archived in Zenodo (https://doi.org/10.5281/zenodo.6462786). RNA co-IP and knockdown mTAIL-seq raw data are available in separate archives in Zenodo (https://doi.org/10.5281/zenodo.6479127 for RNA co-IP, and https://doi.org/10.5281/zenodo.5263366 for knockdown experiments). The mass spectrometry proteomics data are available via ProteomeXchange (PXD028326). Source data are provided with this paper.

Code availability

Custom codes are publicly available online (https://github.com/johapark/polya).

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Acknowledgements

We thank our laboratory members for their support, especially S. Lee for Tet-On inducible TOP TRICK reporter cell line construction and Y. Jung for insights into the inference of deadenylation rates. We also thank J. Kim and J.-S. Kim for mass spectrometry and K. Schönig and J. A. Chao for kind donation of the master cell line and materials used in our reporter cell line construction. This research was supported by the Institute for Basic Science from the Ministry of Science, ICT and Future Planning of Korea (grant no. IBS-R008-D01 to J.P., M.K., H.Y., K.B., Y.C., Y.-s.L., J.L. and V.N.K.); BK21 research fellowships from the Ministry of Education of Korea (to M.K. and Y.C.); and a grant funded by the Kwanjeong Educational Foundation (to M.K.).

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Author notes

  1. Joha Park

    Present address: Massachusetts Institute of Technology, Cambridge, MA, USA

  2. Hyerim Yi

    Present address: Stanford University School of Medicine, Stanford, CA, USA

  3. Young-suk Lee

    Present address: Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea

  4. Jaechul Lim

    Present address: Yale School of Medicine, New Haven, CT, USA

  5. These authors contributed equally: Joha Park, Myeonghwan Kim, Hyerim Yi, Kyungmin Baeg.

Authors and Affiliations

  1. Center for RNA Research, Institute for Basic Science, Seoul, Korea

    Joha Park, Myeonghwan Kim, Hyerim Yi, Kyungmin Baeg, Yongkuk Choi, Young-suk Lee, Jaechul Lim & V. Narry Kim

  2. School of Biological Sciences, Seoul National University, Seoul, Korea

    Joha Park, Myeonghwan Kim, Hyerim Yi, Yongkuk Choi, Jaechul Lim & V. Narry Kim

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Contributions

J.P., H.Y., K.B. and V.N.K. conceived the project. J.P., M.K., H.Y. and Y.C. conducted the cell- and sequencing-related experiments. J.P. performed the bioinformatics analysis. K.B. purified the recombinant proteins and performed the in vitro assay. Y.-s.L. analyzed the PAL-seq dataset. J.L. generated the mTAIL-seq library for the decay machinery knockdown samples. J.P., M.K., H.Y., K.B. and V.N.K. wrote the paper.

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Correspondence to V. Narry Kim.

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Extended data

Extended Data Fig. 1 Simulation to test the relationship between the relative deadenylation rate and the local poly(A) density at a steady state.

a. The production (π) of mRNAs and their poly(A) tails, which includes transcription and 3′ end processing, was modeled with a Gaussian distribution. Deadenylation (δ) and decay (φ) were modeled with logistic functions of the poly(A) length. Deadenylation rates were varied with three different parameters (i)–(iii): uniform, decreasing, and increasing along the poly(A) length (long-to-short), respectively. The number of transcripts over the cycles is shown on the third column to ensure that the system reaches a steady state. The light green-to-black lines in the plots in the right-most column represent the poly(A) length distribution over the cycles. b. Another computer simulation with a different production model. The initial distribution of poly(A) length in the production (π) step is modeled with the negative binomial distribution to account for a more realistic scenario based on the experimental observation34.

Extended Data Fig. 2 Validation experiments after knockdown of decay machinery.

a. Schematic of Tet-On inducible TOP TRICK reporter. Reporter mRNAs are only transcribed in the presence of doxycycline. SV40 poly(A) signal on its 3′ UTR was targeted for measuring poly(A)-tail length by Hire-PAT assay. b. Global poly(A) length distributions measured by mTAIL-seq upon depletion of decay factors. c. Poly(A) length distributions of pulse-expressed reporter mRNAs measured by Hire-PAT assay upon depletion of decay factors at various time points post-wash. d. Steady-state poly(A) length distribution of endogenous mRNAs measured by Hire-PAT assay upon depletion of decay factors.

Extended Data Fig. 3 LARPs as PABPC1 interacting partners and validation experiments evaluating LARPs.

a. Log2 fold of LFQ intensities for anti-PABPC1 over anti-Myc co-immunoprecipitates. The proteins with > 4 folds in both replicates are highlighted and shown in a separate panel. The direct target protein PABPC1 is colored in red, and the co-purified cytosolic RNA-binding proteins are colored in blue. b. The domain structures of LARP1, 4, and 4B. PABP-interacting domains are colored in red. c. Western blotting of cytosol RIP-BPA samples (Exp. 3) in Fig. 2a, b. d. Western blotting of knockdown-BPA samples (Exp. 2) in Fig. 2c, d. e. Poly(A) length distribution of the LARP4, LARP4B single knockdowns and LARP4/4B double knockdown RNAs measured by BPA. f. Western blotting of translational machinery including PABPC1 upon LARP1 knockdown (n = 4).

Source data

Extended Data Fig. 4 Validation experiments for LARP1’s effect on TOP and non-TOP mRNAs.

a. Schematics of the TOP and non-TOP reporter mRNAs. Five cytosines including cytidine on +1 position were replaced with purines58. CTE on their 3′ UTR was targeted for measuring their expression level by RT-qPCR. b. Comparison of LARP1 association with TOP or non-TOP reporter mRNAs. Left: the experimental scheme. Doxycycline was treated for 72 hrs to induce expressions of TOP or non-TOP reporters and reach a steady-state. Center: RT-qPCR of LARP1 co-immunoprecipitated RNAs relative to U1 snRNA (n = 1). The TOP and non-TOP reporter mRNAs were targeted by CTE in their 3′ UTR. GAPDH mRNA was used as a negative control for LARP1 association. Right: Western blotting of the input and LARP1-immunoprecipitated samples. Normal rabbit IgG (NRG) was used as a negative control for immunoprecipitation. c. Poly(A) length distributions of pulse-expressed TOP (left) and non-TOP (right) reporter mRNAs measured by Hire-PAT assay upon depletion of LARP1 using another siRNA (siLARP1 #2). The time indicates the minutes post-wash. d. Steady-state poly(A) length distribution of endogenous mRNAs measured by Hire-PAT assay upon depletion of LARP1 using another siRNA (siLARP1 #2). e. Steady-state poly(A) length distribution of endogenous mRNAs measured by Hire-PAT assay in LARP1-depleted HEK293T and HCT116 cells. f. Abundance changes of the same endogenous mRNAs in (e) relative to U1 snRNA measured by RT-qPCR in LARP1-depleted HEK293T and HCT116 cells (n = 5). Data are represented as mean ± SEM. P-values (*p < 0.05, **p < 0.01, ***p < 0.001) were calculated from the one-sided Student’s t-test.

Source data

Extended Data Fig. 5 Steady-state poly(A) length distribution and mRNA abundance changes upon LARPs knockdown.

a. Global poly(A) length distributions of TOP (left) and non-TOP (right) genes in the LARP1 RNA co-IP sample determined by mTAIL-seq. mt-mRNAs were excluded. b.Global poly(A) length distributions of TOP (left) and non-TOP (right) genes in the LARP1 knockdown sample determined by mTAIL-seq. mt-mRNAs were excluded. c. Poly(A) length distributions of mt-mRNAs determined by mTAIL-seq upon depletion of LARP1 or LARP4/4B. d. Global poly(A) length distributions determined by mTAIL-seq upon depletion of LARP4/4B. mt-mRNAs were excluded. The right plot is scaled by the total abundance of mRNAs relative to mt-mRNAs. e. RT-qPCR of the steadily-expressed TOP reporter relative to U1 snRNA upon LARP1 or LARP4/4B depletion (n = 3). The reporter mRNA was targeted by the common constitutive transport element (CTE) in its 3′ UTR. Data are represented as mean ± SEM. P-values (*p < 0.05) were calculated from one-sided Student’s t-test. f. RT-qPCR of the endogenous mRNAs relative to U1 snRNA upon depletion of LARP4/4B measured by qRT-PCR (n = 4). Data are represented as mean ± SEM. P-values (*p < 0.05) were calculated from the one-sided Student’s t-test.

Source data

Extended Data Fig. 6 In vitro experiments with LARP1, PABPC1 and the CCR-CAF1 subcomplex.

a. InstantBlue staining of purified recombinant proteins (n = 1). b. Double-filter binding assay with PABPC1 and RNAs bearing various lengths of poly(A) tail (left) or LARP1 and the RNAs (right). The mean values ± SE from three independent experiments are shown in the graphs. ‘Kd’ and ‘n.d’. denote ‘dissociation constant’ and ‘not determined’, respectively. c. Analytical size-exclusion chromatography with the purified CCR4-CAF1 subcomplex (C) and LARP1-PABPC1-A25 RNA (LPR). Absorbance at 260 nm and 280 nm were drawn in dashed (A260) or solid (A280) lines. d. In vitro deadenylation assay in the presence or absence of PABPC1 and an increasing amount of LARP1. The A50 substrates were pre-incubated with or without PABPC1 and LARP1 prior to adding the CCR4-CAF1 subcomplex (n = 1). e. Time course in vitro deadenylation assay in the presence or absence of recombinant LARP1 using longer poly(A)-tail substrates (A54, A110). The RNA substrates were pre-incubated with PABPC1 and LARP1 prior to adding the CCR4-CAF1 subcomplex (n = 1).

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Supplementary information

Supplementary Information

Supplementary Table 1: Oligonucleotides used in this study.

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Source data

Source Data Fig. 2

Raw gel images from bulk poly(A)-tail assay.

Source Data Fig. 3

RT–qPCR data.

Source Data Fig. 5

Uncropped gel images for Fig. 5a,b.

Source Data Fig. 5

Size-exclusion chromatography (SEC) data.

Source Data Extended Data Fig. 3

Raw gel images from western blotting and bulk poly(A)-tail assay.

Source Data Extended Data Fig. 4

RT–qPCR data.

Source Data Extended Data Fig. 4

Raw gel images from western blotting.

Source Data Extended Data Fig. 5

RT–qPCR data.

Source Data Extended Data Fig. 6

Raw data from double-filter binding assay and SEC.

Source Data Extended Data Fig. 6

Raw gel/blot images.

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Park, J., Kim, M., Yi, H. et al. Short poly(A) tails are protected from deadenylation by the LARP1–PABP complex. Nat Struct Mol Biol 30, 330–338 (2023). https://doi.org/10.1038/s41594-023-00930-y

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