A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal.

Abstract

Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.Precursors to mRNA are found in the nucleus associated with proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes (reviewed in references 18 and 31). The formation of hnRNP particles occurs immediately after transcription (19,21,37). Many RNA processing events, including splicing and polyadenylation, most likely occur within these complexes and not on naked RNA. Experiments using hnRNP-specific antibodies to inhibit RNA splicing (11, 55), UV-cross-linking analysis (42, 70), and immunoprecipitation of nuclease-resistant RNA segments (61) clearly document an association of hnRNP proteins with splicing and polyadenylation substrate RNAs in vitro. The hnRNP particle, therefore, plays some role in pre-mRNA metabolism.Purified 30S-40S hnRNP particles consist of six major proteins and 20 to 30 auxiliary proteins (6,8,10,15,20,49,68,69). The A1/A2, B1/B2, and C1/C2 species comprise the major hnRNP proteins. Approximately 25 additional proteins are found associated with immunopurified hnRNP particles prepared with antibodies specific for several independent hnRNP proteins (10,19,47) and by two-dimensional gel electrophoresis (69). Many individual hnRNP proteins possess inherent nucleic acid-binding properties (12,30,47,60).The Al protein is a basic, evolutionarily conserved protein with an amino-terminal domain that contains two RNP consensus sequences (1, 41, 50) and a glycine-rich COOHterminal region (12, 13, 30). The Al protein binds nucleic acids cooperatively and may possess helix-destabilizing properties. The hnRNP C proteins are evolutionarily conserved, antigenically related, acidic phosphoproteins that also contain an RNP consensus sequence as well as a putative nucleoside triphosphate-binding site (9, 19, 29, 40, * Corresponding author.48, 62). Numerous multimeric complexes of the A and C proteins, as well as the other hnRNP proteins, have been described (4, 32). C proteins, for example,...

The Poly(A) Tail Inhibits the Assembly of a 3′-to-5′ Exonuclease in an In Vitro RNA Stability System

“…HindIII digestion generated a template which produced the SV or SVA0 RNA. The construction of pSVE-G, which contains the 3Ј UTR of the SV40 early region followed by a polylinker sequence containing a 14-base G-rich sequence, was as described previously for a UUUUU insertion (41). HindIII-linearized plasmid yielded a 171-base RNA following in vitro transcription [SVE-G Poly(A) Ϫ ].…”

Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal.

“…Examination of many polyadenylation signals has shown that besides the conserved AAUAAA sequence, there are surrounding RNA sequences required for efficient utilization of the AAU-AAA as a polyadenylation signal. Downstream efficiency elements (DSEs) have been identified in many different viral systems (Gil andProudfoot 1984, 1987;McDevitt et al 1984McDevitt et al , 1986Sadofsky and Alwine 1984;Cole and Stacy 1985;Conway and Wickens 1985;Sadofsky et al 1985;Zhang and Cole 1987;Wilusz et al 1988;Wilusz and Shenk 1990) at distances from 5 to 60 nucleotides downstream of the poly(A) addition site. These 'Coiiesponding author. elements do not conform to a consensus sequence but are characterized as either U-rich or GU-rich.…”

Common double- and single-stranded DNA binding factor for a sterol regulatory element.

“…There have been reports of sequence-specific binding of single-stranded nucleic acid sequences by heterogeneous nuclear ribonucleoprotein (16,17). The complexes of SRE-BF and single-stranded cognate sequences formed in 6 tug of CHO nuclear extract were found to be resistant to digestion with 1 kug of pancreatic RNase but were completely digested by 10 ng of trypsin.…”