ABSTRACT
Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.
Subject(s)
RNA Precursors , RNA Splice Sites , Animals , Mice , RNA Splice Sites/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , Ribonucleoprotein, U1 Small Nuclear/genetics , RNA Interference , RNA Splicing , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , Alternative Splicing , Mammals/geneticsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.
Subject(s)
Apoptosis Regulatory Proteins/physiology , DNA-Binding Proteins/physiology , Exoribonucleases/physiology , Mouse Embryonic Stem Cells/metabolism , Protein Biosynthesis , RNA Helicases/physiology , RNA Stability , RNA, Messenger/metabolism , Animals , CRISPR-Cas Systems , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Open Reading Frames , Proto-Oncogene Proteins/physiology , RNA, Messenger/chemistry , RNA, Messenger/genetics , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
Eukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5' caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export, and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined during 3' end formation. Most lncRNAs are targeted for nuclear RNA surveillance, but a subset with 3' cleavage and polyadenylation features resembling the mRNA consensus can be exported to the cytoplasm. The Hrp1 and Nab2 proteins act at this decision point, with dual roles in mRNA cleavage/polyadenylation and lncRNA surveillance. Our data also reveal the dynamic and heterogeneous nature of mRNA maturation, and highlight a subset of "lncRNA-like" mRNAs regulated by the nuclear surveillance machinery.
