Identification and Characterization of a Nuclease Specific for the 3′ End of the U6 Small Nuclear RNA

Abstract

The U6 small nuclear RNA is post-transcriptionally processed by the addition and removal of nontemplate uridylates (Us) at its 3 end prior to incorporation into the U4⅐U6 small nuclear ribonucleoprotein complex. An enzyme responsible for removing Us from the U6 3 end has not been previously identified. Here we biochemically isolate and characterize an exonuclease activity from HeLa cells that removes template and nontemplate 3 -nucleotides specifically from U6 RNA. We also report the isolation of an inhibitor of this U6 nuclease. U6 nuclease rapidly removes the four terminal 3 Us found in the human U6 coding sequence in a magnesium-dependent manner. Mutagenesis studies on the U6 RNA define regions essential for processing. U6 nuclease recognizes specific sequences on both strands near the base of the major intramolecular stem loop of the U6 RNA. The preprocessed 3 Us form part of the base of the stem loop, but neither specific sequences nor secondary structure at the four terminal nucleotides are required to achieve processing.RNA polymerase (pol) 1 III produces numerous small RNAs that have a variety of functions (1, 2). pol III transcripts include tRNAs, adenovirus VA 1 RNA, U6 snRNA, 5 S rRNA, and other RNAs such as B1-Alu, which might be involved in retrotransposition of short interspersed elements. One common feature of all these RNAs is a tract of uridylates (Us) at their 3Ј ends that signals pol III to terminate transcription. Apart from pol III termination, these oligo(U) tracts have no known function, although the B1-Alu tail might hybridize with an internal A tract to provide a primer for reverse transcriptase (3)(4)(5).In a number of documented cases, large portions of the 3Ј end of certain pol III transcripts are removed during the course of transcription by the pol III transcription complex (6 -8). Binding of the nuclear autoantigen La protein to the oligo(U) tract of the nascent transcript appears to stabilize it against processing. In other reports of shortened transcripts, the last few nucleotides are absent, which might be due to pol III pausing (9). In this case, the La protein promotes proper termination and transcript release (9).The U6 snRNA transcript undergoes major 3Ј end processing in vivo before it is incorporated into the U4⅐U6 snRNP splicing complex, and the 3Ј region is essential for splicing function (10). At the completion of U6 synthesis, the La protein associates with the 3Ј uridylate tract of U6 (UUUU -OH ) as long as it contains a 3Ј-OH (11). What role the La protein serves in binding to the released transcript is not known, although it may stabilize U6 against degradation. The 3Ј end of La-associated U6 becomes heterogeneous through nontemplate 3Ј addition of Us (11-13). What happens next is unclear; however, the 3Ј end is eventually processed back to five Us in which the terminal U contains a 2Ј,3Ј-cyclic phosphate (UUUU(U)ϾP) (14). The activity responsible for the generation of the 2Ј,3Ј-cyclic phosphate has not been biochemically identified. Formation of the 2Ј,3Ј-cyclic ph...

Effect of 3′ terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes

“…In this study, we isolated a U6 snRNA clone where several 39 uridylic acid residues and one adenylic acid residue encoded by the gene have been removed and the 39 end of U6 snRNA rebuilt by uridylation+ It is unlikely that this U6 snRNA with U at position 102 has arisen from a U6 snRNA gene which contains T corresponding to position 102, as all known human U6 snRNA genes contain A at position 102 of U6 snRNA (Kunkel et al+, 1986;Tichelaar et al+, 1998)+ These data show that, irrespective of the sequence encoded in the gene, the rebuilding in the case of U6 snRNA is accomplished by uridylation+ The most interesting observation made in this study is the high specificity that the uridylating enzyme exhibits in adding uridylic acid residues to 39 U OH -containing RNAs while not adding any U residues to 39 A OH -containing RNAs+ This specificity has been found in vivo as well in the in vitro system+ Several uridylating enzyme activities have been characterized; however, this kind of substrate specificity to 39 U OH -containing RNAs has not been demonstrated in vitro (Zabel et al+, 1981;Andrews et al+, 1985)+ The uridylating enzyme that adds uridylic acid residues is designated terminal uridyl transferase (TUTase), and whether there is a common TUTase acting on the 39 end of many small RNAs or there are RNA-specific TUTases as suggested by Benecke and his associates (Trippe et al+, 1998), is not known+ Isolation and characterization of the uridylating enzyme(s) will help in answering this question+ A multiprotein complex containing several exonucleases, designated exosome, has been characterized in human and yeast cells (Mitchell et al+, 1997)+ This exosome is involved in the formation of accurate 39 ends of many cellular RNAs including small RNAs and it is also responsible for the eventual degradation of RNAs+ The trimming of longer 39 ends that occurs on the 39 ends of many RNAs appears to be carried out by the exosome complex (Allmang et al+, 1999a(Allmang et al+, , 1999b)+ In addition, 39 exonuclease exhibiting specificity to U6 snRNA has been reported (Booth & Pugh, 1997)+ Although the 39 end of cellular RNAs are constantly degraded by complexes like exosome or other nucleases, the adenylation and uridylation activities repair this damage by rebuilding the RNA 39 ends+ Thus, adenylation and uridylation that we characterized in this study and in our earlier reports will be functionally the opposite of exosome function+ The balance between these two events, namely the exosome/nucleases on one side and adenylation/ uridylation on the other side may determine the metabolic fate of the cellular RNAs+ Approximately 65% of the human U2 and SRP RNA molecules contain posttranscriptional 39 adenylation )+ We tested the small RNAs corresponding to SRP and U2 snRNAs from yeast S. cerevisiae for the presence of posttranscriptional adenylation+ Only a low percentage (2-4%) of the yeast SRP or U2 snRNA molecules contained posttranscriptional adenylation; there was no detectable adenylation of yeast 5S rRNA (our unpubl+ data)+ These data are reminiscent of other RNA processing reactions between yeast and higher eukaryotes+ Although only a few yeast genes contain intervening sequences, most genes in higher eukaryotes contain them+ Therefore, the presence of posttranscriptional adenylation and uridylation on the 39 ends of many more RNAs and to a greater extent in higher eukaryotes strongly suggest that these posttranscriptional events m...…”

“…32 P]-UTP+ Lane 1: S-100 extract; lanes 2-4: S-100 extract treated with micrococcal nuclease+ The 5S rRNA with different nucleotides in position 121 added to different tubes are shown on top of each lane+ All other details are as described in Figure 2A+ C: Analysis of the 39 uridylated U6 and 5S RNAs+ The 39 uridylated U6 snRNA RNA from Figure 4A, lane 3, and 39 uridylated 5S RNA from Figure 4B, lane 4, were isolated, purified, and digested to completion by T2 RNase+ The digestion products of U6 snRNA (left panel) and 5S RNA (right panel) were fractionated by twodimensional chromatography and subjected to autoradiography+ The unlabeled nucleotides visualized under UV light are shown as standard mononucleotide markers+ extensive posttranscriptional uridylation on the 39 end of U6 snRNA (Rinke & Steitz, 1985;Reddy et al+, 1987;Terns et al+, 1992;Gu et al+, 1997)+ The 39 processing is a dynamic process where uridylation and deuridylation occur simultaneously (Gu et al+, 1997)+ Booth andPugh (1997) reported the presence of a nuclease specific for the 39 end of U6 snRNA+ One possible function of the 39 uridylation is to rebuild 39 ends that are shortened by 39 exonucleases+ If true, the sequence that is rebuilt by uridylation will contain only U residues no matter what the sequence was in the primary transcript+ The data obtained in the case of human U6 snRNA is consistent with this hypothesis+ The nucleotide corresponding to position 102 in the human U6 snRNA gene and in the RNA primary transcript is A (see Table 1 and Fig+ 6, right panel)+ However, in one interesting clone out of nearly 100 human U6 snRNA clones sequenced, the nucleotide corresponding to position 102 is uridylic acid instead of A (see Fig+ 6, left panel)+ These data show that trimming is not confined to the 39 uridylic acid residues and rebuilding of the 39 end is only by uridylation, no matter what the nucleotide was in the primary RNA transcript+ Therefore, detection of U6 snRNA transcripts in which adenylic acid residue corresponding to position 102 has been replaced with uridylic acid strongly supports the possibility that one of the functions of 39 uridylation is to rebuild the 39 ends trimmed by exonucleases+ All previously published data and the results presented in this study are supportive of a mechanism where two competing processes work on the 39 end of small RNAs+ Uridylation is one process that adds uridylic acid residues to 39 U OH -containing small RNAs; this has been shown to be true for U6 snRNA and 5S rRNA in vivo and in vitro+ Adenylation is the other process where a single adenylic acid residue is added to the 39 end; this has been shown to occur in vivo on the 39 ends of SRP, 7SK, U2, 5S, and U6 snRNAs+ However, the 39 A OH -containing RNAs cannot be uridylated+ Thus, the extent of 39 adenylation modulates the 39 uridylation+ These data are supportive of a hypothetical model that is summarized and presented in Figure 7+ As shown in this figure, uridylation and deuridylation are reversible; similarly, adenylation and deadenylation are also reversible+ Although 39 U OH -containing RNAs can be adenylated, 39 A OH -containing RNAs cannot be uridylated+…”

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

“…Whether the 3Ј box functions as a transcription termination signal or as an RNA processing signal is still unknown (25); however, the first detectable U snRNA precursors have 3Ј-terminal tails that extend nearly to the 3Ј box (22,31,32,35,36,71) and are trimmed by cytoplasmic nucleases that may be related to the yeast multinuclease complex known as the exosome (10,35,36,38).…”

“…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).…”

Isolation and Characterization of a U-specific 3′-5′-Exonuclease from Mitochondria of Leishmania tarentolae

“…An exonuclease activity specific for removal of the post-transcriptionally added (5) 3Ј-oligo(U) tail of U6 small nuclear RNA has been partially purified and characterized (6). A number of 3Ј-5Ј-exoribonucleases have also been described in bacteria and shown to be involved in RNA processing.…”