Formation of the 3'' end of U1 snRNA is directed by a conserved sequence located downstream of the coding region.

U1 is a small non-polyadenylated nuclear RNA that is transcribed by RNA polymerase II and is known to play a role in mRNA splicing. The mature 3' end of U1 snRNA is formed in at least two steps. The first step generates precursors of U1 RNA with a few extra nucleotides at the 3' end; in the second step, these precursors are shortened to mature U1 RNA. Here, I have determined the sequences required for the first step. Human U1 genes with various deletions and substitutions near the 3' end of the coding region were constructed and introduced into HeLa cells by DNA transfection. The structure of the RNA synthesized during transient expression of the exogenous U1 gene was analyzed by S1 mapping. The results show that a 13 nucleotide sequence located downstream from the U1 coding region and conserved among U1, U2 and U3 genes of different species is the only sequence required to direct the first step in the formation of the 3' end of U1 snRNA.     

Nucleotide sequence at the 5'' end of ovalbumin messenger RNA from chicken.

DNA-sequence analysis of 300 nucleotides from the region of cloned, double-stranded ovalbumin cDNA corresponding to the 5' end of ovalbumin messenger RNA was accomplished using the technique of Maxam and Gilbert (Proc. Nat. Acad. Sci. USA (1977) 74,560-564). The AUG initiation codon was located 52 nucleotides from the AT linkers used in cloning and immediately adjacent to the amino terminal peptide of ovalbumin, indicating the absence of a "signal peptide" in this protein. The nucleotide sequence coding for a phosphorylated peptide from ovalbumin was also found. These results demonstrate that the coding portion of mRNAov begins near the 5' end of the molecule leaving some 600 nucleotides of noncoding information at the 3' end.     

The 5''-terminal sequence of U1 RNA complementary to the consensus 5'' splice site of hnRNA is single-stranded in intact U1 snRNP particles.

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.     

Point mutations in the stem-loop at the 3'' end of mouse histone mRNA reduce expression by reducing the efficiency of 3'' end formation.

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.     

Control of cleavage site selection during mRNA 3'' end formation by a yeast hnRNP.

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.     

A novel role of U1 snRNP: Splice site selection from a distance

Removal of introns by pre-mRNA splicing is fundamental to gene function in eukaryotes. However, understanding the mechanism by which exon-intron boundaries are defined remains a challenging endeavor. Published reports support that the recruitment of U1 snRNP at the 5′ss marked by GU dinucleotides defines the 5′ss as well as facilitates 3′ss recognition through cross-exon interactions. However, exceptions to this rule exist as U1 snRNP recruited away from the 5′ss retains the capability to define the splice site, where the cleavage takes place. Independent reports employing exon 7 of Survival Motor Neuron (SMN) genes suggest a long-distance effect of U1 snRNP on splice site selection upon U1 snRNP recruitment at target sequences with or without GU dinucleotides. These findings underscore that sequences distinct from the 5′ss may also impact exon definition if U1 snRNP is recruited to them through partial complementarity with the U1 snRNA. In this review we discuss the expanded role of U1 snRNP in splice-site selection due to U1 ability to be recruited at more sites than predicted solely based on GU dinucleotides.     

The 5'' end group of tobacco mosaic virus RNA is m7G5'' ppp5'' Gp.

RNA extracted from CsC1-purified virions of tobacco mosaic virus is shown to give rise to an unusual nucleotide on digestion which RNAase T2, in addition to the four major nucleotides. This minor component has the electrophoretic characteristics of a phosphorylated end group, but is partially resistant to bacterial alkaline phosphatase. It is, however, a substrate for venom phosphodiesterase or nucleotide pyrophosphatase, yielding products which imply the structure m7G5'ppp5'Gp. TMV RNA, like many animal cellular and viral mRNAs recently examined, therefore has a 5' terminus blocked by a methylated nucleotide inverted with respect to the rest of the chain.     

The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing.