Intrinsic Disorder and the Evolution of Overlapping Genes

THE TEAM:Pedro R. Romero1, Corinne Rancurel2, Mahvash Khosravi1,

Keith Dunker1, and David Karlin3

1Indiana University - Purdue University Indianapolis, Indianapolis, IN, USA, 2Architecture et Fonction des Macromolécules Biologiques, Campus de Luminy, Marseille,

France3Tous Chercheurs, Inmed, Parc de Luminy, 13273 Marseille Cedex 09, France

Overlapping Genes and “Overprinting”

• Overlapping genes discovered in first sequenced genome (Phage Φ-X174, Sanger, 1977)

• Many theoretical studies in the ’70s– Constrained evolution demonstrated

– Limited information content/gene (Yockey, 78)

– Predicted difference in evolutionary speed (Smith & Waterman, 78)

• “Overprinting” of older genes proposed as mechanism for de novo gene generation (Keese & Gibbs, 1992)

• Structural effects noticed in isolated examples: some recent ones shown to be related to intrinsic disorder.

Overprinting: Creation of a novel C-terminal extension

Overprinting Examples

Overprinting and Structure Example: Measles virus (Karlin, et al, 2002)

Figure 1. A schematic view of the measles virus. The nucleocapsid protein (N) assembles into a cylindrical capsid that wraps the virus’ RNA. The nucleocapsid protein has a disordered tail where the phosphoprotein (P) binds. The P protein is largely disordered, and it is encoded by a multiple-encoding area of the RNA (see Figure 2).

Figure 2. Schematic view of the RNA region that encodes the P, V, and C proteins in the measles virus genome. The color lines at the bottom of the diagram represent the three RNA reading frames. The phosphoprotein (P) and the N-terminus of the V protein are encoded on frame 1 (blue), the C-terminus of the V protein is encoded on frame 2 (red), and the entire C protein is encoded on frame 3 (green). The colored cylinders at the top represent the encoded protein products, with the narrow cylinders denoting disordered regions and the wide cylinders representing ordered regions. Notice that no region in the RNA encodes two ordered protein domains.

Intrinsically Disordered Proteins

• IDPs and regions are less sensitive to evolutionary changes than ordered ones

• They undergo faster evolution than ordered proteins or regions

• More dependent on residue content than position

• Mostly participants in regulatory and signaling functions

Structural analysis of viral overlapping genes

Hypotheses1. Intrinsic disorder might help alleviate

evolutionary constraints in overlapping genes

2. New, overprinted genes will likely tend to be more disordered than ancestral counterpart

Structural analysis of viral overlapping genes

• Need for real, expressed genes• Spliced genes excluded (many

unannotated splicing events)

• Assembled data from 43 viral genomes– Unspliced RNA virus– Unspliced retroid virus (RNA and DNA)– Overlaps > 90 nucleotides (30 residues)

Analysis of viral overlapping genes

Analysis of viral overlapping genesTable 2. Predicted order/disorder statistics on overlapping genes data set.

Confidence Intervals Measures of order content Fraction 68% (1 std. dev.) 95%

Fraction of sequence predicted ordered Entire data set 71% 68-74% 66-76% Encoded by overlapping genes 52% 48-57% 45-60% Fraction of overlapping sequence positions predicted ordered on both protein products

Expected 50% 46-55% 44-58% Observed 28% 23-33% 19-36%

0

0.1

0.2

0.3

0.4

0.5

0.6

Expected Observed

Frac

tionO

-O

Difference in disorder contentbetween entire data set andoverlapping regions is significant(p-value 3x10-57)

Difference between expected andobserved fraction of order-orderoverlaps is also significant(P-value 5x10-24)

Structural and functional organization of ancestral/novel proteins

Structural and functional organization of ancestral/novel proteins

Structural and functional organization of ancestral/novel proteins

• Overprinting (novel) proteins– Most are Orphans (no homologs outside of

genus)– Mostly disordered– Mostly accessory proteins– Proteins created by overprinting different

homologs of the same gene display a wide diversity of functional and of structural features

Disorder and evolutionary constraints

HBV Sendai SIV HTLV phiX174A-B

phiX174D-E

PLRV HPV CLCuVAC1-AC4

CLCuVCP-AV2

0%

20%

40%

60%

80%

100%

% d

isor

der i

n ov

erla

ppin

g re

gion

Virus

More constrainedLess constrained

HBV Sendai SIV HTLV phiX174A-B

phiX174D-E

PLRV HPV CLCuVAC1-AC4

CLCuVCP-AV2

0%

20%

40%

60%

80%

100%

% d

isor

der i

n ov

erla

ppin

g re

gion

Virus

More constrainedLess constrained

Disorder and evolutionary constraints

Disorder and evolutionary constraints

• Experiment shows that less constrained (faster evolving) proteins in a pair tend to be more disordered

• Only exceptions when disorder content very similar between the two overlapping proteins

Conclusions

• Both proposed hypothesis supported:– Disorder appears to alleviate evolutionary

constraints– Novel overprinted genes tend to be

disordered• New directions: Show whether novel

genes tend to be disordered “at birth” or are selected to be disordered

Figure 1. A schematic view of the measles virus. The nucleocapsid protein (N) assembles into a cylindrical capsid that wraps the virus’ RNA. The nucleocapsid protein has a disordered tail where the phosphoprotein (P) binds. The P protein is largely disordered, and it is encoded by a multiple-encoding area of the RNA (see Figure 2).

THANKS!!