A 3′-Terminal Minihelix in the Precursor of Human Spliceosomal U2 Small Nuclear RNA

Abstract

U2 RNA is one of five small nuclear RNAs that participate in the majority of mRNA splicing. In addition to its role in mRNA splicing, the biosynthesis of U2 RNA and three of the other spliceosomal RNAs is itself an intriguing process involving nuclear export followed by 5-cap hypermethylation, assembly with specific proteins, 3 end processing, and then nuclear import. Previous work has identified sequences near the 3 end of pre-U2 RNA that are required for accurate and efficient processing. In this study, we have investigated the structural basis of U2 RNA 3 end processing by chemical and enzymatic probing methods. Our results demonstrate that the 3 end of pre-U2 RNA is a minihelix with an estimated stabilization free energy of ؊6.9 kcal/mol. Parallel RNA structure mapping experiments with mutant pre-U2 RNAs revealed that the presence of this 3 minihelix is itself not required for in vitro 3-processing of pre-U2 RNA, in support of earlier studies implicating internal regions of pre-U2 RNA. Other considerations raise the possibility that this distinctive structural motif at the 3 end of pre-U2 RNA plays a role in the cleavage of the precursor from its longer primary transcript or in its nucleocytoplasmic traffic.The major spliceosome that operates on most eukaryotic pre-mRNAs contains five small RNAs, U1, U2, U4, U5, and U6. Beyond their role in mRNA splicing, the biosynthesis of these small spliceosomal RNAs is itself an interesting process. U6 small nuclear is transcribed by RNA polymerase III (1, 2) and associates with spliceosomes without extensive 3Ј-processing or nucleocytoplasmic transit (3). In contrast, the other four major spliceosomal RNAs, U1, U2, U4, and U5, are transcribed by RNA polymerase II in mammalian cells as precursor molecules extended at their 3Ј ends that are then exported to the cytoplasm where they undergo cap hypermethylation and ribonucleoprotein assembly followed by 3Ј end processing and nuclear import (4 -13).The mammalian precursor molecules of U1, U2, U4, and U5 RNAs have been defined in several studies (4, 6, 8 -11, 14), and their 3Ј-processing has been well characterized particularly for U2 RNA (11,13,[15][16][17]. A distinct region of pre-U2 RNA lying near the 3Ј end of mature RNA has been shown to be critical for accurate and efficient 3Ј-processing (16). It was also found that sequences in the 5Ј-half of pre-U2 RNA play no role in the 3Ј-processing reaction (16), suggesting that in the folded structure of pre-U2 RNA, the 5Ј-half of the molecule is not interactive with the 3Ј end, at least not in a way that influences the 3Ј-processing reaction. The 3Ј end of pre-U2 RNA also has been implicated in the nucleocytoplasmic traffic of U2 RNA. Nonprocessed 3Ј end variants of pre-U2 RNAs display impaired nuclear import in both Xenopus oocytes (18) and human cells (19), indicating that cytoplasmic 3Ј-processing is a key step in the nuclear import pathway.The 3Ј end of the precursor of human U2 RNA consists of an 11-nucleotide element (11,14,20,21), but the structure of this 3Ј tail is ...

The 3' ends of human pre-snRNAs are produced by RNA polymerase II CTD-dependent RNA processing

“…The U2 3′ box position is indicated by a dashed box. The long arrows above the sequence represent nts included in the stem–loop IV of mature U2 snRNA (Reddy et al ., 1981) and the 3′ terminal minihelix is in grey (Mougin et al ., 2002). The processing sites observed in vivo and in vitro in the presence of the USE are indicated by short arrows.…”

“…Indeed, deletion of this region affects 3′ end formation of human U2 snRNA precursors synthesized in Xenopus oocytes (Yuo et al ., 1985). The USE also contains the 3′ terminal minihelix recently identified in the pre‐U2 snRNA structure and suggested to play a role in 3′ end formation of pre‐U2 snRNA (Mougin et al ., 2002). Interestingly, a computer‐putative secondary structure of the USE contains the minihelix with the processing sites located within an unpaired bulge (Figure 5D).…”

Expression of human snRNA genes from beginning to end

“…The 3 -end formation of human snRNAs transcribed by pol II occurs in several steps initiated by recognition of the cis-acting 3 -box [4]. This 13-16-nt-long element directs the production of a 3 extended pre-snRNA, which is a specific substrate for subsequent processing that generates the mature 3 -end after transport to the cytoplasm [14][15][16].…”