3′ Processing of Human Pre-U2 Small Nuclear RNA: a Base-Pairing Interaction between the 3′ Extension of the Precursor and an Internal Region

Abstract

The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3 ends. The 3 processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3 processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3 ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3 end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3 processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3 extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3 extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3 processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3 processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3 extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3 processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3 processing of human U2 RNA.Small nuclear RNAs (snRNAs) are essential components of ribonucleoprotein particles that are involved in pre-mRNA processing (1, 16). The biosynthetic maturation pathway of the spliceosomal snRNAs involves many steps in both the nucleus and cytoplasm (20,35). Except for U6 (14,26), the snRNAs of the major class of spliceosomes are transcribed by RNA polymerase II. Once transcribed, pre-U1, -U2, -U4, and -U5 snRNAs carrying 3Ј extensions of various lengths are rapidly transported to the cytoplasm, where they undergo small nuclear ribonucleoprotein (snRNP) assembly, 5Ј-cap hypermethylation, and 3Ј processing before being imported back into the nucleus (3, 4, 6, 8, 13, 17-20, 25, 32, 34, 35).U2 RNA is the second most abundant snRNA in mammalian cell nuclei (25). Pre-U2 RNA molecules having 3Ј extensions of 10 to 16 nucleotides have been detected in both rat liver (29) and HeLa cells (7,32). We have previously shown that pre-U2 RNA 3Ј processing occurs accurately in several in vitro systems of progressive refinement, including HeLa cell whole cytoplasmic extract (32), an ammonium sulfate fraction of a HeLa cell cytoplasmic S100 fraction (12), and a 15S glycerol gradient fraction of the latter (9, 12). Because this 15S system is free of a nonspecific ribonuclease present in the former preparations, it has enabled us to study pre-U2 RNA 3Ј processing in considerable detail (9). In co...

U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase)

“…The 3Ј-terminal A of U2 snRNA must be added posttranscriptionally because the U2 snRNA coding region ends with CC/CC, where the slash indicates the last encoded nucleotide (45)(46)(47). If 3Ј exonucleolytic processing removes the encoded 3Ј CC during U2 snRNA maturation, as appears to be the case in vitro (33,34,38), then the cell may need to build or rebuild the 3Ј-terminal A, CA, or CCA of U2 snRNA. Using a variety of synthetic and natural U2 snRNAs as * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…U2ϩ10 is subsequently exported to the cytoplasm, where the 3Ј tail is trimmed (31)(32)(33)(34) by endonuclease and exonuclease activities that could be related to the yeast exosome, a multinuclease complex involved in processing many cellular RNAs (35,36). Sm antigens then assemble onto the Sm binding site, and the resulting immature U2 snRNP is imported into the nucleus for final assembly into a mature U2 snRNP (35,(37)(38)(39).…”

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

“…Human pre-U2 RNA with a wild-type 3Ј tail and lacking nucleotides 1-104 (non-essential for 3Ј-processing) was transcribed from the plasmid pMRG3U2-54 (16). Pre-U2 RNAs with mutant 3Ј tails, U2-75 and U2-80, were transcribed from the plasmids described previously (17). The RNAs were labeled at their 5Ј ends with g-[ 4 dpm/l) were subjected to Pb 2ϩ -mediated cleavage in 50 mM potassium acetate, 10 mM magnesium acetate, 50 mM HEPES-KOH, pH 7.5, containing 4 or 10 mM PbCl 2 .…”

“…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).…”

“…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).…”