2.7.3 Maturation of the Primary Transcripts (RNA Processing)
39
also spliced enzymatically, although the enzymes
involved (endonuclease, ligase, etc.) are not
organized in a splice os orne [63]. Soon after initiation, the 5' ends ofthe primary Pol II transcripts
receive the cap sequence m 7 GpppXpmY ...
(Fig. 2.9); these protect the transcript from attack
by 5' -exonucleases and bind the finished mRNA
onto the large ribosomal subunit. The pre-mRNA
is already linked to proteins during transcription
to form the "string-of-pearls" type of hnRNP particle. The hnRNP particles contain a set of evolutionarily conserved polypeptides, amongst which
are six so-called core proteins of 30-43 kDa
[34, 119, 350, 357]. With the attachment of different, smaller nuclear RNAs (snRNAs, p. 52), the
hnRNP particle is converted into a "spliceosome", on which the excision of the introns and
the ligation of the exons (splicing) takes place
[119]. Upon completion of transcription, excess
nucleotides are removed from the 3' end of the
Pol II transcript, and a poly(A) sequence of initially 200-300 nt is added; the poly(A) tail of
mature mRNA in the cytoplasm is usually shorter.
Some mRNAs, e.g. most histone mRNAs, are
not polyadenylated [264]. The function of the
poly(A) chain is not clear, although it apparently
increases the in vivo stability of the mRNA [281].
Accurate splicing requires particular signals at
the intron-exon border Gunctions), the consensus
sequences of which are AG:GUAAGU at the 5'
end of the intron, and (U/C)uNCAG:G at the 3'
end [208, 336]. Occasionally, one finds signals
deviating from the "GU-AG rule" but which,
nevertheless, function perfectly: e.g. GC- instead
of GU- is found at the 5' end of the second intron
of the aD -globin gene of the duck Cairina moschata [126]. An example of a modified 3' signal
with -CG instead of -AG is provided by the per
gene of Drosophila [77]. The splicing process
begins with a break at the 5' end of the intron.
The exposed 5'-G is linked by a 2',5'phosphodiester bond to an internal A residue
near the 3' end of the intron; this produces a circular structure with a tail (a lariat) (Fig. 2.10).
Just how the intron becomes fully released and
the exons ligated is not completely clear. The
whole process requires ATP. All stages of RNA
maturation take place in the nucleus; the finished
mRNA is then transported into the cytoplasm by
an as yet incompletely understood mechanism,
but it is known that it becomes bound to a further
set of proteins [119].
In the Kinetoplastida, like Trypanosoma,
Leishmania or Crithidia, mRNA maturation does
not involve linking of fragments of the premRNA molecule, but rather the binding of the
pre-mRNA to an independent transcript (transsplicing). Consequently, all mature mRNAs of
each species begin with the same 35- to 39-nt
sequence, the "spliced leader" (SL) or "miniexon". The SL sequences differ according to the
species but are quite similar, e.g. between Crithidia fasiculata and Trypanosoma brucei they agree
at 27 positions (77 % ). In T. brucei, Leptomonas
collosoma and most likely other Kinetoplastida,
the SLs have an unusual terminal cap structure of
pm 7 G ... followed by four 02'-methylnucleotides.
The SLs originate from the 5' end of an SL RNA,
with a length of 85-140 nt, which is encoded by
its own specific gene family. SL or mini-exon
genes have lengths of 0.4-1.35 kb and form
tandem clusters of 200-250 copies. The primary
transcripts of the Kinetoplastida are multicistronic and during maturation are cleaved at
specific sequences into individual protein-coding
pre-mRNAs; these are further processed at the 5'
end by trans-splicing and at the 3' end by attachment of a poly(A) chain. The typical polyadenylation signal AAUAAA is most certainly missing
Fig. 2.10. The mechanism of the splicing process, during
which an intron in the primary transcript is excised and the
breakpoint resealed [394]. In the first step, the GpG bond
at the 5' end of the intron is broken and the exposed 5'terminal G of the intron is linked to an internal A by a 2',
5' -phosphodiester bond. The tailed, circular structure that
results is known as a lariat. In the second step, the 3'terminal OH group of the leading exon is linked to the 5'
phosphate of the succeeding exon and the lariat is released
39
also spliced enzymatically, although the enzymes
involved (endonuclease, ligase, etc.) are not
organized in a splice os orne [63]. Soon after initiation, the 5' ends ofthe primary Pol II transcripts
receive the cap sequence m 7 GpppXpmY ...
(Fig. 2.9); these protect the transcript from attack
by 5' -exonucleases and bind the finished mRNA
onto the large ribosomal subunit. The pre-mRNA
is already linked to proteins during transcription
to form the "string-of-pearls" type of hnRNP particle. The hnRNP particles contain a set of evolutionarily conserved polypeptides, amongst which
are six so-called core proteins of 30-43 kDa
[34, 119, 350, 357]. With the attachment of different, smaller nuclear RNAs (snRNAs, p. 52), the
hnRNP particle is converted into a "spliceosome", on which the excision of the introns and
the ligation of the exons (splicing) takes place
[119]. Upon completion of transcription, excess
nucleotides are removed from the 3' end of the
Pol II transcript, and a poly(A) sequence of initially 200-300 nt is added; the poly(A) tail of
mature mRNA in the cytoplasm is usually shorter.
Some mRNAs, e.g. most histone mRNAs, are
not polyadenylated [264]. The function of the
poly(A) chain is not clear, although it apparently
increases the in vivo stability of the mRNA [281].
Accurate splicing requires particular signals at
the intron-exon border Gunctions), the consensus
sequences of which are AG:GUAAGU at the 5'
end of the intron, and (U/C)uNCAG:G at the 3'
end [208, 336]. Occasionally, one finds signals
deviating from the "GU-AG rule" but which,
nevertheless, function perfectly: e.g. GC- instead
of GU- is found at the 5' end of the second intron
of the aD -globin gene of the duck Cairina moschata [126]. An example of a modified 3' signal
with -CG instead of -AG is provided by the per
gene of Drosophila [77]. The splicing process
begins with a break at the 5' end of the intron.
The exposed 5'-G is linked by a 2',5'phosphodiester bond to an internal A residue
near the 3' end of the intron; this produces a circular structure with a tail (a lariat) (Fig. 2.10).
Just how the intron becomes fully released and
the exons ligated is not completely clear. The
whole process requires ATP. All stages of RNA
maturation take place in the nucleus; the finished
mRNA is then transported into the cytoplasm by
an as yet incompletely understood mechanism,
but it is known that it becomes bound to a further
set of proteins [119].
In the Kinetoplastida, like Trypanosoma,
Leishmania or Crithidia, mRNA maturation does
not involve linking of fragments of the premRNA molecule, but rather the binding of the
pre-mRNA to an independent transcript (transsplicing). Consequently, all mature mRNAs of
each species begin with the same 35- to 39-nt
sequence, the "spliced leader" (SL) or "miniexon". The SL sequences differ according to the
species but are quite similar, e.g. between Crithidia fasiculata and Trypanosoma brucei they agree
at 27 positions (77 % ). In T. brucei, Leptomonas
collosoma and most likely other Kinetoplastida,
the SLs have an unusual terminal cap structure of
pm 7 G ... followed by four 02'-methylnucleotides.
The SLs originate from the 5' end of an SL RNA,
with a length of 85-140 nt, which is encoded by
its own specific gene family. SL or mini-exon
genes have lengths of 0.4-1.35 kb and form
tandem clusters of 200-250 copies. The primary
transcripts of the Kinetoplastida are multicistronic and during maturation are cleaved at
specific sequences into individual protein-coding
pre-mRNAs; these are further processed at the 5'
end by trans-splicing and at the 3' end by attachment of a poly(A) chain. The typical polyadenylation signal AAUAAA is most certainly missing
Fig. 2.10. The mechanism of the splicing process, during
which an intron in the primary transcript is excised and the
breakpoint resealed [394]. In the first step, the GpG bond
at the 5' end of the intron is broken and the exposed 5'terminal G of the intron is linked to an internal A by a 2',
5' -phosphodiester bond. The tailed, circular structure that
results is known as a lariat. In the second step, the 3'terminal OH group of the leading exon is linked to the 5'
phosphate of the succeeding exon and the lariat is released