ABSTRACT
The 5' terminal sequence of U1 snRNA that base-pairs with the intron 5' splice site in the course of spliceosome assembly was considered to be universally conserved. A study of the P polycephalum U1 snRNA at both RNA and gene levels shows that there are exceptions to this rule: the P polycephalum U1 snRNA has a U to A substitution at position 5, that is partially compensated by a high frequency of T residue at position +4 of introns. In contrast to the yeast genome, the P polycephalum genome contains several U1 snRNA coding sequences (about 20). They either encode the U1A snRNA expressed in microplasmodia or correspond to the previously cloned U1B coding sequence. Both coding sequences show the U5A substitution. The ratio of U1A versus U1B coding sequences is of about 3. A U1A gene was cloned. The 60 nt region upstream of the coding sequence has the same sequence as in the U1B gene. The U1B gene is probably expressed at another stage of the P polycephalum life cycle.
Subject(s)
Physarum polycephalum/chemistry , RNA, Small Nuclear/chemistry , Amino Acid Sequence , Animals , Blotting, Southern , Cloning, Molecular , DNA Primers , DNA, Fungal/chemistry , DNA, Fungal/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression/genetics , Introns/genetics , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA Splicing/genetics , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Small Nuclear/genetics , Sequence Alignment , Sequence AnalysisABSTRACT
In order to study the phylogenetic conservation of modified nucleotides in the spliceosomal U5 snRNA, we determined the nucleotide sequences of the U5 snRNAs from the slime mold Physarum polycephalum (EMBL data bank accession numbers: X74440 and X74441) and we identified the pseudouridine and 2'-O-methylated residues. From a comparison of all the U5 snRNAs studied at the level of nucleotide modifications, we concluded that the modified nucleotides in U5 snRNA can be divided into three classes according to their degree of conservation: i) the modified nucleotides of the 5' terminal cap structure that display some variations from one species to the other; ii) the modified nucleotides located in the helical part of the stem/loop structure I that vary greatly in number, position and identity from one species to the other; and iii) the modified nucleotides of the terminal loop 1, that are almost identical in all the species studied. Taking into account the recent discovery of a crucial role played by this terminal loop of U5 snRNA in 5' and 3' splice site definition, we postulate that the numerous modified nucleotides it contains, five out of a total of 11, play an important role in spliceosome assembly and function. Their possible role is discussed.
