Streamlined and sensitive mono- and diribosome profiling in yeast and human cells

Ferguson, Lucas; Upton, Heather; Pimentel, Sydney C.; Mok, Amanda; Lareau, Liana F.; Collins, Kathleen; Ingolia, Nicholas T.

doi:10.1101/2023.02.01.526718

Cited by 6 publications

(15 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resulting over- and under-representation of certain footprints leads to distorted ribosome distributions, which can be detected as artifactual signals showing an influence of several nucleotides at either end of footprints on footprint recovery [13, 15, 16]. New ligation-free methods such as the template jumping approach used in OTTR ribosome profiling greatly ameliorate sequence preferences, but cannot completely avoid them [49, 50]. Removing these sequence signals is thus necessary to reveal accurate and precise patterns from ribosome profiling data and to uncover mechanisms of translation regulation and their biological functions.…”

Section: Discussionmentioning

confidence: 99%

choros: correction of sequence-based biases for accurate quantification of ribosome profiling data

Mok

Tunney

Benegas

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Ribosome profiling quantifies translation genome-wide by sequencing ribosome-protected fragments, or footprints. Its single-codon resolution allows identification of translation regulation, such as ribosome stalls or pauses, on individual genes. However, enzyme preferences during library preparation lead to pervasive sequence artifacts that obscure translation dynamics. Widespread over- and under-representation of ribosome footprints can dominate local footprint densities and skew estimates of elongation rates by up to five fold. To address these biases and uncover true patterns of translation, we present choros, a computational method that models ribosome footprint distributions to provide bias-corrected footprint counts. choros uses negative binomial regression to accurately estimate two sets of parameters: (i) biological contributions from codon-specific translation elongation rates; and (ii) technical contributions from nuclease digestion and ligation efficiencies. We use these parameter estimates to generate bias correction factors that eliminate sequence artifacts. Applying choros to multiple ribosome profiling datasets, we are able to accurately quantify and attenuate ligation biases to provide more faithful measurements of ribosome distribution. We show that a pattern interpreted as pervasive ribosome pausing near the beginning of coding regions is likely to arise from technical biases. Incorporating choros into standard analysis pipelines will improve biological discovery from measurements of translation.

show abstract

Section: Discussionmentioning

confidence: 99%

choros: correction of sequence-based biases for accurate quantification of ribosome profiling data

Mok

Tunney

Benegas

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition to the simplicity and low bias of OTTR-seq, the low input requirement of the protocol also allows better and easier sRNA sequencing of low-RNA-yield samples such as extracellular vesicles and mature sperm [ 57 , 62 ]. PNK treatment can be added to this protocol to further enrich sRNA classes, as discussed above [ 62 , 63 ].…”

Section: Current Methods For Sequencing Srnasmentioning

confidence: 99%

Sperm RNA Payload: Implications for Intergenerational Epigenetic Inheritance

Liu

Sharma

2023

IJMS

View full text Add to dashboard Cite

There is mounting evidence that ancestral life experiences and environment can influence phenotypes in descendants. The parental environment regulates offspring phenotypes potentially via modulating epigenetic marks in the gametes. Here, we review examples of across-generational inheritance of paternal environmental effects and the current understanding of the role of small RNAs in such inheritance. We discuss recent advances in revealing the small RNA payload of sperm and how environmental conditions modulate sperm small RNAs. Further, we discuss the potential mechanism of inheritance of paternal environmental effects by focusing on sperm small RNA-mediated regulation of early embryonic gene expression and its role in influencing offspring phenotypes.

show abstract

“…To test whether eIF3 engagement at 3'-UTR termini correlates with translation activity at a transcriptome-wide level, we performed ribosome profiling, a technique that is based on deep sequencing of ribosome-protected mRNA fragments (RPFs) 23 , in undifferentiated and differentiated NPCs. Here, we used a low-bias ribosome profiling approach that takes advantage of the enzymatic activities of reverse transcriptase (RT) from eukaryotic retroelements to reduce the technicalities associated with ribosome profiling 23,24 . In addition, this method uses P1 nuclease instead of the commonly used RNase I and replaces RNA ligation with Ordered Two-Template Relay (OTTR) 24 .…”

Section: Eif3 Engages With 3'-utr Termini Upon Increased Levels Of Pr...mentioning

confidence: 99%

eIF3 engages with 3’-UTR termini of highly translated mRNAs

Mestre-Fos,

Ferguson,

Trinidad

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 (ID2) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs.

show abstract

Streamlined and sensitive mono- and diribosome profiling in yeast and human cells

Cited by 6 publications

References 52 publications

choros: correction of sequence-based biases for accurate quantification of ribosome profiling data

choros: correction of sequence-based biases for accurate quantification of ribosome profiling data

Sperm RNA Payload: Implications for Intergenerational Epigenetic Inheritance

eIF3 engages with 3’-UTR termini of highly translated mRNAs

Contact Info

Product

Resources

About