Format

Send to

Choose Destination
RNA. 2015 Nov;21(11):1943-65. doi: 10.1261/rna.053389.115. Epub 2015 Sep 16.

A multiprotein occupancy map of the mRNP on the 3' end of histone mRNAs.

Author information

1
Department of Genetics, Dartmouth Geisel School of Medicine, Hanover, New Hampshire 03755, USA.
2
Integrative Program for Biological and Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
3
Department of Computer Science, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

Abstract

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP.

KEYWORDS:

3′hExo; CIMS; CLIP-seq; H2A.X; H2AFX; HITS-CLIP; RBPome; RIP-chip; RIP-seq; RNP; SLBP; UPF1; histone mRNA

PMID:
26377992
PMCID:
PMC4604434
DOI:
10.1261/rna.053389.115
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center