Abstract
In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3′ end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3′ end processing of histone pre-mRNAs.
Highlights
Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that are not polyadenylated, ending instead with a conserved stem-loop followed by a short single-stranded tail of 4–5 nucleotides [1,2]
Our studies show that the semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition as U7 snRNP purified from nuclear extracts and is active in 3 end processing of histone pre-mRNAs, functionally mimicking endogenous U7 snRNP
SmB lacked the unstructured C-terminal region located between amino acids 96 and 231 that was likely to affect alignment of the U7 snRNP particles in electron microscopy
Summary
Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that are not polyadenylated, ending instead with a conserved stem-loop followed by a short single-stranded tail of 4–5 nucleotides [1,2] They are formed from longer mRNA precursors (pre-mRNAs) by a single endonucleolytic cleavage carried out by U7 snRNP, a metazoan-specific minor snRNP that is ∼500fold less abundant than the major spliceosomal snRNPs. Its RNA component, U7 snRNA, is the shortest known snRNA (∼60 nucleotides) and consists of three functionally distinct regions [3,4,5,6]. The 5 end region of ∼15 nucleotides base pairs with the sequence in histone pre-mRNA known as Histone Downstream Element (HDE) This region is primarily responsible for the substrate specificity of U7 snRNP for histone pre-mRNAs. The 9-nucleotide AAUUUGUCU sequence located immediately downstream of the 5 end region is referred to as the Sm binding site [7]. The Sm binding site in U7 snRNA is followed by an extensive 3 stem-loop that in vivo may facilitate the assembly of the Sm ring and protect U7 snRNA against the activity of 3 exonucleases
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