Embedded transfer RNA Genes
Genes
(a), Smarajit Das ( a ), Jayprokas Chakrabarti ( a ,
Zhumur Ghosh (a)
Bibekanand Mallick ( a ) and
b,* )
Satyabrata Sahoo ( a )
(a) Computational Biology Group (CBG)
Theory Department
Indian Association for the Cultivation of Science
Calcutta 700032 India
(b) Biogyan
BF 286, Salt Lake
Calcutta 700064 India
* Author for correspondence
Telephone: +91-33-24734971, ext. 281 (Off.)
Fax: +91-33-24732805
E-mail: [email protected]
Abstract: In euryarchaeal methanogen M.kandleri and in Nanoarchaea N.
equitans some of the missing tRNA genes are embedded in others. We argue from
bioinformatic evidence that position specific intron splicing is the key behind colocation of these tRNA genes.
Key words:
words embedded tRNAs, archaea, intron, splicing.
1
Introduction:
In our recent work1 we analysed cytoplasmic tRNA genes ( tDNA ) of 22 species of
12 orders of three phyla of archaea. We looked for the identity elements for
aminoacylation. During this investigation we found some tDNAs missing in
euryarchaea and nanoarchaea. We observed later that some of these missing
tDNAs lie embedded in other tDNAs. In this communication we argue that
bioinformatic evidence points towards intron splicing at alternate positions in
these embedded tDNAs. One composite tDNA gives rise to two different tRNAs.
The single-stranded primary tRNA nucleotide chain folds back onto itself
to form the cloverleaf secondary structure. This structure has: (i) Acceptor or A
arm: In this the 5/ and 3/ ends of tRNA are base-paired into a stem of 7 bp. (ii)
DHU or D arm: Structurally a stem-loop, D-arm frequently contains the modified
base dihydrouracil. (iii) Anticodon or AC arm, made of a stem and a loop
containing the anticodon. At 5/ end of this anticodon-loop is a pyrimidine base at
32, followed by an invariant U at 33. The anticodon triplet is located at 34, 35, and
36 in the exposed loop region. (iv) An Extra Arm or V arm: This arm is not always
present. It is of variable length and largely responsible for the variation in lengths
of tRNAs. tRNA classification into types I and II depend on length of V-arm . (v) TΨ-C arm or T arm: This has conserved sequence of three ribonucleotides:
ribothymidine, pseudouridine and cytosine. T arm has stem-loop structure and (vi)
tRNA terminates with CCA at 3/ end. tDNA may or may not have CCA; If absent
,it is added during tRNA maturation.
Introns were found in several archaeal tDNAs between tRNA-nucleotide-positions
37 and 38, located in AC–loop2. These are the canonical introns (CI). Archaea, an
intermediate between Eukarya and Bacteria, have tRNAs that share many
similarities with either or both these domains3. Archaeal tDNAs harbour introns
at various locations other than the canonical position of tRNA. These are the
noncanonical introns (NCI).
2
Although, noncanonical introns in tDNAs were observed in 19874,
bioinformatic identification of tDNAs harbouring these continues to be a
challenge. As we developed our algorithm to circumvent some of the difficulties,
we found evidence that tRNA genes overlap in archaea through introns, canonical
and noncanonical. Earlier, in mitochondrial tRNAs, overlaps of between one to six
nucleotides have been reported. tRNATyr and tRNACys genes in human
mitochondrial genome, for instance5 , overlap with one another by one nucleotide (
the last base of tRNACys and discriminator base of tRNATyr ). But tDNA-overlaps
in archaea is an altogether new phenomena. In euryarchaeal methanogen
Methanopyrus kandleri6 AV19 (NC_003551) and in nanoarchaea Nanoarchaeum
equitans Kin4-M
7
(NC_0005213) we find entire tDNAs embedded within one
another.
Introns are present most frequently at the canonical position 37/38 in ACloop. Apart from these, introns are also located in AC-arm, V-arm, D-arm and Tarm as well in A-arm1.
The exact location of archaeal introns is obtained by
looking for the presence of the bulge-helix-bulge (BHB) motif8. Archaeal splicing
machinery cleaves introns at variable positions in tDNAs within the BHB motif9.
In archaea, the tRNA endonuclease plays a key role in the removal of the intron
from pre-tRNAs10. Hence, splicing of introns is a RNA-protein interaction which
requires mutual recognition of two complementary tertiary structures.
Methodology
The tRNA search programs like tRNAscan-SE and ARAGORN key on primary
sequence patterns and/or secondary structures specific to tRNAs. A few loop-holes
exist in these algorithms. It is the inability of these existing routines to identify
tRNA genes if it harbours noncanonical introns in them.
Some tRNAs are
misidentified; some are missed out. We developed a computational approach to
search for tDNAs that have noncanonical introns. With this algorithm we
identified some non-annotated tDNAs. About one thousand tRNA-genes from
archaea were studied for this purpose. From this database of 1000 tRNA-genes we
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fine-tuned the strategy to locate non-canonical introns. The salient features were
:(i) sequences
were considered that gave rise to the regular cloverleaf secondary
structure. (ii ) conserved elements : T8 (except Y8 in M. kandleri), G18, R19, R53,
T44, Y55, and A58 were considered as conserved bases for all archaeal tRNA .
Further there were tRNA-specific conserved or identity elements1 of archaea. (iii)
the constraints of lengths of stems of regular tRNA A-arm, D-arm, AC-arm and Tarm were 7, 4, 5 and 5 bp respectively. In few cases the constraints on lengths of
D-arm and AC-arm were relaxed.
(iv)Promoter sequence ahead of the 5/-end
looked for. ( v ) Base positions optionally occupied in D-loop were 17, 17a, 20a and
20b. (vi) Extra arm or V-arm was considered for tRNAs. The constraint on length
of V-arm: less than 21 bases (vi) Noncanonical introns were considered at any
position. The introns constrained to harbour the Bulge-Helix-Bulge (BHB)
secondary structure for splicing out during tRNA maturation. The minimum
length of introns allowed was 6 bases.
Results and Conclusions:
tRNAGly / tRNAeMet Embedded Genes of M. kandleri
This is our first example of embedded tRNA genes. In fig 1 we illustrate this
embedding of two tDNAs.
tRNAGly(CCC) gene remained unidentified in M. kandleri. Note this gene is
present in other archaea . Using our algorithm we identify it between c382165 and
382053. The sequence is presented is figure 1. This glycine tRNA has the
important bases A73, C35 and C36 necessary for aminoacylation by AARS
(aminoacyl tRNA synthetase)11. It has the conserved bases and base-pairs of other
archaeal glycine tRNAs. In this tDNA, presumably12, the 15 base long intron
located at 32/33 is processed before splicing of second intron, 21 bases long, at
37/38. It has consensus BHB motif of type hebh/bh/L (shown in figure 2). This
sequential removal of introns implies that there is enough plasticity of tRNA
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molecule within the whole AC-stem and loop to allow major rearrangements
between two successive splicing process .
One of the elongator methionine tRNA gene lies exactly embedded in this range.
This eMet tRNA gene has all the important features of archaeal elongator
methionine tRNA. C34, A35 and U36 are the identity elements in addition to the
discriminator base A73 in this tRNA. It has a canonical intron of length 36. Part of
the same BHB structure but this intron has a different splice-site marked in
figure 2. The 3/ -splice-site for the canonical introns of the two embedded tRNA
genes is the same .
tRNAGlu / tRNAeMet Embedded Genes of N. equitans
This is the second example. Note in fig 1 we illustrate these embedded tRNA
genes.
In N. equitans all the tRNA genes could not be located using tRNAscan-SE. Some
of the missing ones were later found from the split-tRNA hypothesis13,14 . We
identify tRNAGlu(CUC) in this genome lying between bases 327362 and 327500 of
the genome.
It has two introns, one canonical and one noncanonical. The
canonical intron is 26 bases long. The noncanonical intron is located between
bases 31 and 32 of AC-loop. The length of this noncanonical intron is 40 bases. The
conserved bases and bps of archaeal tRNAGlu are consistent in this tRNA as well.
U35 and C36 are identity elements for archaeal Glu tRNA as in E. coli
C5:G68 could be another identity element1 for archaeal tRNAGlu .
15,16.
All these
identity elements are well present in this embedded tRNAGlu gene. The entire
intronic structure has heB[(h1/ L1) (h2/ L2) (h3/ b h3/ L3)] type BHB motif and has
proper splice-sites ( figure 3).
The elongator methionine tRNA17 gene also lies within this range. This
tRNA has C34, A35 and U36 as the identity elements in addition to the
discriminator base A73. These features are consistent with all other archaeal
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elongator methionine tRNA. It has a canonical intron of length 66. This also has
the same BHB, albeit with different splicing position, marked in fig 3. The
canonical introns of the embedded tRNA genes, once again, have the same 3/ splice site.
In some of the primary transcripts of mitochondrial tRNA of animals,
tRNAs are known to overlap by one to several bases
18.
In archaea we find tRNAs
fully embedded in one other. The release of the entire versions of the two
embedded tRNAs is assumed to occur . We believe one of the tRNAs is correctly
processed in some transcripts, the other in other transcripts, potentially producing
both complete transcripts. In these possibilities, the mode of recognition between
the primary transcript and the processing enzyme(s) remains unclear. Presumably
there exist sequence/structural patterns within the precursor tRNA, upstream or
downstream, encoding this embedding. We are investigating features of pre-tRNA
responsible for alternate endonucleolytic splicing of introns.
Acknowledgements:
We acknowledge useful discussions with Chanchal Dasgupta and Siddhartha Roy.
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Figure 2
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Figure 3
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Figure Legends
Figure 1: Sequences of the embedded tRNA genes.
Blue coloured region denotes noncanonical intron, brown canonical intron and
black the exonic region.
Figure 2: Secondary structure of tRNAGly(CCC) / tRNAeMet(CAT) along with
the BHB of their introns of M. kandleri.
NCIs: Noncanonical intron start position; NCIe: Noncanonical intron end position;
CIs: Canonical intron start position; CIe: Canonical intron end position
This signifies splicing sites of the introns in pre-tRNAs
he: Exonic helix; h/ : mixed helix (part of it is intronic and part of it is exonic)
b: bulge; L: loop
Figure 3: Secondary structure of tRNAGlu(CTC) / tRNAeMet(CAT) along with
the BHB of their introns of N. equitans.
NCIs: Noncanonical intron start position; NCIe: Noncanonical intron end position;
CIs: Canonical intron start position; CIe: Canonical intron end position
This signifies splicing sites of the introns in pre-tRNAs
he: Exonic helix; h/: mixed helix (h1/ : mixed helix in the 1st branch; h2/ : mixed
helix in the 2nd branch; h3/ : mixed helix in the 3rd branch )
L : loop (L1: loop in the 1st branch; L2 : loop in the 2nd branch; L3 : loop in the 3rd
branch )
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