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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.

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S.A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25 degrees C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25 degrees C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations.

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.