Applications of Polysome Sequencing Across Biomedical and Plant Research

Precise regulation of gene expression is a core mechanism of life activities, and translation efficiency, as a key link in post-transcriptional regulation, directly affects protein synthesis rate and functional diversity. Traditional research has focused primarily on the transcriptional level, but systematic explanations of core questions such as how mRNA is recognized by ribosomes and how translation rate is dynamically regulated remain lacking. Polysome sequencing (Polysome-seq), by isolating mRNAs loaded on different ribosomes, has for the first time achieved single-molecule-level quantification of translational activity, providing a new perspective for revealing disease mechanisms, crop stress resistance, and biological evolution. In recent years, groundbreaking discoveries in areas such as cancer metabolic reprogramming and plant abiotic stress responses have highlighted its dual value in basic research and practical applications.

This article systematically reviews the technical principles and cutting-edge cases of Polysome-seq, aiming to promote its in-depth application in interdisciplinary research.

Technical Principles and Advantages

Polysome-seq, based on sucrose density gradient centrifugation, utilizes the high sedimentation coefficient of ribosomes to separate mRNAs loaded with different ribosomes: free RNA, single ribosomes (80S), and polyribosomes (mRNAs bound to multiple ribosomes). Quantitative analysis of each component via RNA sequencing allows for precise assessment of translation efficiency and detection of translational activity in non-coding regions (such as the UTR) and non-classical RNAs (such as lncRNA and circRNA).

Technical Advantages

• "Gold Standard" for Translation Efficiency: Directly reflects mRNA translational activity, superior to other indirect methods.
• Dynamic Monitoring Capability: Captures translational changes under stress, drug treatment, and other conditions, such as increased ribosome binding to mRNA under oxidative stress.
• Multi-Dimensional Integration: Can be combined with RNA-seq and epitranscriptomics (such as m7G modification) for joint analysis, elucidating translational regulatory networks.

Applications in Biomedical Research

(1) Cancer Mechanism Analysis

Chen Y et al., through polysome profiling, showed that in MVD KD cells, the GPX4 mRNA content was reduced in the high multigranule group (groups 13-19, representing actively translated mRNA-ribosome complexes), but the total mRNA level did not decrease.

This result confirms the inhibitory effect of MVD KD on GPX4 translation—although the total amount of GPX4 mRNA remained unchanged, the amount of actively translated mRNA (multigranule-bound state) decreased, indicating that MVD promotes the translation of selenoproteins (such as GPX4) by maintaining tRNA modifications (such as i⁶A37 tRNA modification), while MVD deficiency leads to translation arrest, providing translational evidence for the subsequent conclusion that "MVD inhibits induced ferroptosis."

MVD promoted CoQ10 synthesis and selenocysteine-tRNA modification (Chen Y et al., 2025)

Du D et al. used polysome sequencing, m⁷G tRNA MeRIP-Seq, and Ribo-seq technologies to reveal the key mechanism of METTL1-m⁷G tRNA modification-translation regulation in BC, providing new targets for BC treatment (such as combined CDK4/6 inhibitors):

• METTL1 regulates the translation of BC-related genes (such as GADD45A and RB1) in a codon-dependent manner.
• The reduced levels of METTL1-regulated polynucleotide-related mRNAs (such as GADD45A and RB1) confirm its translational repression effect.
• Combined with Ribo-seq, it was clarified that METTL1 selectively blocks translation during the G2/M phase of the cell cycle through m⁷G tRNA modification.
• By increasing tRNA m⁷G levels, the translation of key genes such as GADD45A and RB1 is promoted, blocking the G2/M cell cycle progression and inhibiting BC proliferation.

METTL1 enhances m7G tRNA methylation levels and global mRNA translation (Du D et al., 2024)

To learn more about the applications of polyribosomal sequencing in cancer research, please refer to "Applications of Polysome Sequencing in Cancer Research".

(2) Neurological and Metabolic Diseases

Chak K et al., using polysome sequencing, qRT-PCR, and Northern blot techniques, jointly discovered the competitive mechanism between the pre-miR-21 and mature miR-21, revealing its molecular basis for regulating TGFBR2/NT-3 in epilepsy:

• Pre-miR-21 and mature miR-21 compete for binding sites on the 3'UTR of TGFBR2 mRNA (overlapping energy advantages), but do not compete for NT-3 mRNA;
• After pre-miR-21 binds to the TGFBR2 3'UTR, it counteracts the degradation/translational inhibition of TGFBR2 by mature miR-21, leading to increased TGFBR2 expression and decreased NT-3 expression after a seizure (SE);
• Polysome Sequencing analysis revealed that pre-miR-21 is located in the multisomal component of active translation, suggesting that it may counteract the inhibition of miR-21 posttranscribedly, prolonging TGF-β receptor expression and affecting epilepsy.
• The ratio of pre-miR-21 to mature miR-21 is a core factor regulating the translational repression/degradation of certain mRNAs (such as TGFBR2 and E2f6). An imbalance in the pre-/mature ratio may lead to disease progression (such as epilepsy).

Pre-miR-21 is associated with mRNAs engaged in active translation (Chak K et al., 2016)

To learn more about the applications of polyribosomal sequencing in neuroscience, please refer to "Polysome Sequencing in Neuroscience: Insights into Brain Translation".

Li Q et al., using polysome profiling combined with RNA stability analysis, protein stability analysis, and RIP techniques, discovered that YTHDF1 promotes hypoxia-induced autophagy and its progression in HCC through m⁶A-dependent translational regulation. Their findings are as follows:

• YTHDF1 knockout: ATG2A/ATG14 mRNA shifts from the "multimer (active translation component)" to the "non-multimer," resulting in decreased mRNA content in the translation component → translational repression;
• YTHDF1 overexpression: ATG2A/ATG14 mRNA shifts to the "multimer," resulting in increased mRNA content in the translation component → translational activation.
• YTHDF1-WT (wild-type) can immunoprecipitate ATG2A/ATG14 mRNA, while YTHDF1-MUT (m⁶A binding pocket mutation) cannot. YTHDF1 binds to target mRNA through the m⁶A binding pocket.
• YTHDF1 promotes the expression of autophagy-related genes ATG2A/ATG14 through m⁶A-dependent translational activation, thereby driving hypoxia-induced autophagy, growth, and metastasis in HCC.

Polysome profiling of hypoxic SMMC7721 and Hep3B cells (Li Q et al., 2021)

To learn more about the applications of polyribosomal sequencing in immunology, please refer to "Polysome Sequencing in Immunology: Translational Regulation of Immune Responses".

Applications in plant research

(1) Abiotic Stress Response

Juntawong P et al., using polysome profiling combined with immunopurification (IP), qRT-PCR, and confocal imaging, discovered the association between the Arabidopsis cold shock protein CSP1 (RNA-binding protein RBP) and ribosomes, and its selective translational regulation under stress conditions. Their findings were as follows:

• CSP1 co-localization with ribosomes: The epitope-labeled CSP1-FLAG (35S:AtCSP1-FH#11 transgene) was only present in the multimeric component (active translation complex containing ≥2 ribosomes), and not in the low-density monomer or 40S subunit;
• Global translation was unaffected: The multi-body levels in transgenic seedlings were similar to those in wild-type Col-0, indicating that CSP1 overexpression does not alter overall protein synthesis;
• Changes under cold stress: After 4°C cold treatment, the abundance and multi-body co-localization of CSP1 increased, but the global multi-body level was not affected (multi-body levels are insensitive to temperatures above freezing).
• CSP1 is a selective translation regulator that binds to multimers (actively translating mRNA-ribosome complexes) and specific mRNAs (rich in G+C 5'UTR and involved in cellular respiration/translation). Under cold stress or water deficiency, CSP1 abundance and multimer colocalization are enhanced, promoting the translation of these mRNAs and thus enhancing plant stress resistance. Its action is selective (does not affect global translation) and depends on its association with ribosomes.

CSP1 is localized in the nucleus and cytosol, and co-purifies with polysomes (Juntawong P et al., 2013)

(2) Fruit Ripening Regulation

Polysome profiling was used to study the effect of SlYTH2 (tomato m⁶A reading protein) knockout on fruit translation efficiency. The results showed that:

• SlYTH2 knockout (slyth2 mutant) did not change the levels of 40S or 60S ribosomal subunits, but the level of 80S monomers (single ribosomes) decreased significantly.
• Starting from segment 8 (representing multi-ribosomes, i.e., active translation complexes with ≥2 ribosomes), the number of dimers, trimers, and multimers in the slyth2 mutant increased significantly.
• The increase in multimer peaks (active translation complexes) was accompanied by a decrease in 80S monomers (inactive ribosomes), indicating that the translation efficiency of slyth2 mutant fruits was superior to that of wild-type (WT).
• Polysome profiling technology directly revealed the regulatory role of SlYTH2 in translation efficiency: SlYTH2 reduces the translation efficiency of target genes (SlHPL, SlCCD1B) by inhibiting the binding of multinucleotide polysomes; after knocking out SlYTH2, the binding of multinucleotide polysomes of target genes increases, translation efficiency is improved, and ultimately leads to an increase in aromatic volatiles and enhanced fruit aroma.

SlYTH2 inhibits translation efficiency of SlHPL and SlCCD1B, thereby suppressing protein accumulation (Bian H et al., 2024)

Technical Comparison and Limitations

Key Limitations

• High Sample Input: Requires ≥1×10⁷ cells, limiting application for low-abundance or rare samples.
• Lack of Single-Cell Resolution: Current protocols are not yet adapted for single-cell analysis, relying instead on population-level profiling.

Future Development Directions

• Single-cell polyribosome analysis: Developing micro-sample processing techniques to analyze cellular heterogeneity.
• Multi-omics integration: Combining m6A-seq and CRISPR screening to construct translation regulatory networks (such as the autophagy axis in cancer).
• Dynamic tracking technology: Utilizing time-series experiments to reveal transient changes in translation events, such as rapid regulation in stress responses.

Conclusion: Polysome-seq, by quantifying translation efficiency and ribosome distribution, provides an irreplaceable tool for research on disease mechanisms, crop improvement, and environmental adaptation. With the advancement of single-cell technology and multi-omics integration, it will play an even greater role in precision medicine and agricultural biotechnology.

References

1. Chen Y, Lee D, Kwan KK, Wu M, Wang G, Zhang MS, Deng H, Cheu JW, Lau MH, Chan CY, Ooi ZY, Wu Y, Bao MH, Lo RC, Ng IO, Wong CM, Wong CC. Mevalonate pathway promotes liver cancer by suppressing ferroptosis through CoQ10 production and selenocysteine-tRNA modification. J Hepatol. 2025 Dec;83(6):1338-1352.
2. Du D, Zhou M, Ju C, Yin J, Wang C, Xu X, Yang Y, Li Y, Cui L, Wang Z, Lei Y, Li H, He F, He J. METTL1-mediated tRNA m7G methylation and translational dysfunction restricts breast cancer tumorigenesis by fueling cell cycle blockade. J Exp Clin Cancer Res. 2024 May 31;43(1):154.
3. Chak K, Roy-Chaudhuri B, Kim HK, Kemp KC, Porter BE, Kay MA. Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation. Exp Neurol. 2016 Dec;286:137-146.
4. Li Q, Ni Y, Zhang L, Jiang R, Xu J, Yang H, Hu Y, Qiu J, Pu L, Tang J, Wang X. HIF-1α-induced expression of m6A reader YTHDF1 drives hypoxia-induced autophagy and malignancy of hepatocellular carcinoma by promoting ATG2A and ATG14 translation. Signal Transduct Target Ther. 2021 Feb 23;6(1):76.
5. Juntawong P, Sorenson R, Bailey-Serres J. Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana. Plant J. 2013 Jun;74(6):1016-28.
6. Bian H, Song P, Gao Y, Deng Z, Huang C, Yu L, Wang H, Ye B, Cai Z, Pan Y, Wang F, Liu J, Gao X, Chen K, Jia G, Klee HJ, Zhang B. The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process. Proc Natl Acad Sci U S A. 2024 Jul 9;121(28):e2405100121.