3' processing of human pre-U2 small nuclear RNA: a base-pairing interaction between the 3' extension of the precursor and an internal region.

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.     

Sequences required for 3' end formation of human U2 small nuclear RNA

Xenopus oocytes injected with human U2 snRNA genes synthesize mature U2 as well as a U2 precursor with about 10 extra 3' nucleotides (human pre-U2 RNA). Formation of the pre-U2 3' end requires a downstream element located between position +16 and +37 in the U2 3'-flanking sequence. The distance between this element and the U2 coding region can be increased without affecting formation of the pre-U2 3' end. When the natural sequence surrounding the pre-U2 3' end is changed, novel 3' ends are still generated within a narrow range upstream from the element. The 3' terminal stem-loop of U2 snRNA is not required for pre-U2 3' end formation. A sequence within the 3' element (GTTTN0-3AAAPuNNAGA) is conserved among snRNA genes transcribed by RNA polymerase II. Our results suggest that the 3' ends of pre-U2 RNA and histone mRNA may be generated by related but distinct RNA processing mechanisms.     

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.     

The role of intron structures in trans-splicing and cap 4 formation for the Leishmania spliced leader RNA.

A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae spliced leader RNA 5' modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental to trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastid Leptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3' end processing. The critical nature of the L. tarentolae Sm-binding site is consistent with its essential role in the Ascaris spliced leader RNA, whereas in Leptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway for Leishmania spliced leader RNA processing and maturation is proposed.     

Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides

Yeast U2 snRNA (1175 nucleotides) is six times larger than its mammalian counterpart (188 nucleotides). Using deletion analysis, we show that the molecule can be divided into three phenotypically distinct domains. As expected, the highly conserved 5' domain (approximately 120 nucleotides) is absolutely essential for viability. Surprisingly, however, deletion of the central 945 nucleotides has no effect on growth rate. In contrast, removal of sequences in the 3' terminal 110 nucleotides results in low numbers of slow-growing colonies; these cells contain U2 with altered 3' ends. This domain can be folded into a secondary structure that strongly resembles the 3' terminal stem-loop IV of human U2. We conclude that yeast U2 contains two functionally important elements. While the 5' domain is known to be directly involved in the splicing reaction, the 3' domain may function primarily in the generation of stable small nuclear ribonucleoprotein particles.     

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro . To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro .     

An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.     

Nuclear processing of the 3''-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes.

U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end. We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus. The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor. This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein. TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA. The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures. TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions. We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA.     

Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure.

Human immunodeficiency virus type 1 Rev protein modulates the distribution of viral mRNAs from the nucleus to the cytoplasm by interaction with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To identify the minimal functional elements of RRE, we evaluated mutant RREs for Rev binding in vitro and Rev response in vivo in the context of a Gag expression plasmid. The critical functional elements fold into a structure composed of a stem-loop A, formed by the ends of the RRE, joined to a branched stem-loop B/B1/B2, between bases 49 and 113. The 5' 132 nucleotides of RRE, RREDDE, which possessed a similar structure, bound Rev efficiently but were nonfunctional in vivo, implying separate binding and functional domains within the RRE. Excision of stem-loop A reduced Rev binding significantly and abolished the in vivo Rev response. The B2 branch could be removed without severe impairment of binding, but deletions in the B1 branch significantly reduced binding and function. However, deletion of 12 nucleotides, including the 5' strand of stem B, abolished both binding and function, while excision of the 3' strand of stem B only reduced them. Maintenance of the native RRE secondary structure alone was not sufficient for Rev recognition. Many mutations that altered the primary structure of the critical region while preserving the original RNA conformation were Rev responsive. However, mutations that changed a 5'..CACUAUGGG..3' sequence in the B stem, without affecting the overall structure abolished both in vitro Rev binding and the in vivo Rev response.     

Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies.

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.     

3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.     

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.     

A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import.

The biosynthesis of U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs) involves the nuclear export of precursor molecules extended at their 3' ends, followed by a cytoplasmic phase during which the pre-snRNAs assemble into ribonucleoprotein particles and undergo hypermethylation of their 5' caps and 3' end processing prior to nuclear import. Previous studies have demonstrated that the assembly of pre-snRNAs into ribonucleoprotein particles containing the Sm core proteins is essential for nuclear import in mammalian cells but that 5' cap hypermethylation is not. In the present investigation we have asked whether or not 3' end processing is required for nuclear import of U2 RNA. We designed human pre-U2 RNAs that carried modified 3' tails, and identified one that was stalled (or greatly slowed) in 3' end processing, leading to its accumulation in the cytoplasm of human cells. Nonetheless, this 3' processing arrested pre-U2 RNA molecule was found to undergo cytoplasmic assembly into Sm protein-containing complexes to the same extent as normal pre-U2 RNA. The Sm protein-associated, unprocessed mutant pre-U2 RNA was not observed in the nuclear fraction. Using an assay based on suppression of a genetically blocked SV40 pre-mRNA splicing pathway, we found that the 3' processing deficient U2 RNA was significantly reduced in its ability to rescue splicing, consistent with its impaired nuclear import.