RNA Binding by Sxl Proteins in Vitro and in Vivo

Abstract

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

RNA Secondary Structure: An Important cis-Element in Rat Calcitonin/CGRP Pre-Messenger RNA Splicing

“…Our results indicate that cleavages by the double-strand-specific RNase CV 1 were consistently strong within the region that makes up the polypyrimidine tract (Figure 4, nucleotides [19][20][21][22][23][24][25][26][27][28][29][30]. These results indicate that the polypyrimidine tract could be recognized inefficiently due to two factors (35,36). First, this region contains several purines, which have been shown to reduce the affinity for U2AF 65 , an important component in early spliceosome formation (36).…”

“…Strong CV1 cleavages were always observed near the noncanonical branchpoint, nucleotides 10-16. The polypyrimidine tract of the splice acceptor is interrupted by several purines, making it a poor match to the mammalian consensus sequence (34)(35)(36). Our results indicate that cleavages by the double-strand-specific RNase CV 1 were consistently strong within the region that makes up the polypyrimidine tract (Figure 4, nucleotides [19][20][21][22][23][24][25][26][27][28][29][30].…”

“…These results indicate that the molecule is essentially double-stranded, and also that the overall tertiary structure may keep the splice signals located in the intron accessible. This is important because trans-acting molecules needed for spliceosome formation, such as U2AF 65 /U2AF 35 , polypyrimidine binding protein (36), and U2 snRNP, would still have access to their sites of interaction with the pre-mRNA.…”

Contributions of the RNA-Binding and Linker Domains and RNA Structure to the Specificity and Affinity of the Nucleolin RBD12/NRE Interaction

“…RNA Synthesis and Purification. Unlabeled and uniformly 13 C, 15 N-labeled RNA oligonucleotides for NMR studies were prepared by in vitro transcription as described (41), except for the single-stranded substrates, which were chemically synthesized (Dharmacon, Boulder, CO) and purified as previously described (41). b2NRE and sNRE were annealed at dilute concentrations (1-10 µM) in water and adjusted to the desired salt conditions by the addition of the appropriate stock solution.…”

“…Protein-RNA complexes were prepared by addition of aliquots of lyophilized RNA or protein until the indicated molar ratio was achieved. Titration of nucleolin RBD12 and RBD12 subdomains into 0.15 mM 13 C, 15 N-labeled sNRE or b2NRE was monitored at 310 K by gradient sensitivity enhanced 1 H- 13 C heteronuclear single-quantum coherences (HSQCs) (45). Titration of SSNRE or SSNS into 0.15 mM 15 N-labeled nucleolin RBD12 was monitored at 310 K by 1 H- 15 N HSQCs with WATERGATE (46) water suppression.…”